Occurrence of Human Norovirus GII and Human Enterovirus in Ontario Source Waters

Cassandra Diane LoFranco

A Thesis Presented to The University of Guelph

In partial fulfilment of requirements for the degree of Master of Science in Environmental Biology

Guelph, Ontario, Canada

ABSTRACT

Occurrence of Human Norovirus and Human Enterovirus in Ontario Source Waters

Cassandra Diane LoFranco Advisors: H. Lee University of Guelph, 2017 M. Habash S. Weir

Norovirus and Enterovirus are common human viral pathogens found in water sources. Despite causing gastroenteritis outbreaks, most jurisdictions, including Ontario, do not monitor for enteric viruses in waters. The objective of this thesis was to monitor the presence of human

Norovirus and Enterovirus in Ontario source waters intended for drinking.

Two untreated source water types (river and ground water) were sampled routinely and following precipitation and snow melt events between January 2015 and April 2016. Physical, chemical, microbiological, and meteorological data were collected, coinciding with sampling events. A modified USEPA Method 1615 was applied to detect and quantify viruses and logistic regression was used to examine relationships between virus presence and environmental parameters.

Norovirus was detected in 41% of river water and 33% of groundwater samples. Enterovirus was detected in 18% of river water and 29% of groundwater samples. No correlations between virus detection and environmental parameters were found.

Acknowledgements

With tremendous gratitude and appreciation, I’d like to thank the following people:

Professors Hung Lee and Marc Habash who made the call inviting me to this project team and worked closely with me on the project and finer details of this paper and Dr's Susan Weir and Paul Sibley for their time and perspectives. Not only do I appreciate your knowledge and expertise, but your insightful comments and suggestions that most certainly improved the paper, but your enthusiasm and encouragement, which made it possible for me to forge ahead and complete my work. When faced with overwhelming personal and professional challenges throughout this journey, it was your support and confidence in me and my abilities that held me on course to attain this graduate degree, and for this I am forever grateful.

Nicole, my being here today would not be possible without your initiative and interest in water safety and for your success in acquiring grant funding and establishing essential municipal connections necessary to carry out the investigation. Thank you to Tim and his professional staff at the Region of Waterloo, who were most kind and helpful with access to the sampling sites.

My dear friends and seasoned experts: Sonya, Susan, Anne-Marie, Janis, Dae Young, and Rebecca, for always being there for me even when my inner voice and spirit were flailing. I couldn’t have done this without your broad shoulders, and supportive advice – thank you from the bottom of my heart for believing in me and keeping me grounded. For friends and colleagues at the Habash lab and MOECC, who supported my involvement on this project, thank you.

And above all, my wonderful husband Doug and children, Domenic and Luca for their love and support, managing the day to day operations of our home so that I could focus on my research and writing, enabling me to spend long hours tapping away at my keyboard or at the lab running experiments until the early hours, I love you with all my heart.

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And to myself, I am proud to have been the oldest female graduate student (to date) within the SES department to compete the M. Sc., and I am so proud to have accomplished this long-standing personal goal – It was tough at times, but I never gave up!

Dedication

Dedication of this paper is to anyone of any age who keeps within their hearts a personal goal and is looking for hope, encouragement and support that they can make it happen. Never give up on your personal aspirations and never fear trying to attain your goals in the midst of the naysayers and personal and professional challenge. Be tenacious, be persistent; maintain that inner fire burning however tiny, as anything worth having, requires a lot of time, hard work and may require multiple attempts.

To my dad, so dearly missed, - may he rest in peace and my mother for her early teachings on how to be independent and strong ... “Never stop learning”

Table of Contents ABSTRACT ...... 2

Acknowledgements ...... iii

Dedication ...... v

Table of Contents ...... vi

List of Tables ...... x

List of Figures ...... xi

List of Appendices ...... xii

List of Abbreviations ...... xiii

Chapter 1: Introduction and Literature Review ...... 1

1.1 Waterborne disease from human enteric viruses ...... 1

1.2 Norovirus ...... 4

1.2.1 Agent ...... 4

1.2.2 Taxonomy ...... 5

1.2.3 Structure of the genome ...... 5

1.3 Enteroviruses ...... 6

1.3.1 Agent ...... 6

1.3.2 Taxonomy ...... 7

1.4 Seasonality of Norovirus and Enterovirus ...... 8

1.5 Prevalence and persistence of human Norovirus and human Enterovirus in source waters ...... 11

1.5.1 Survival and Inactivation of Norovirus...... 15

1.5.2 Survival and Inactivation of Enteroviruses ...... 16

1.6 Methods for the Collection and Detection of Norovirus and Enterovirus from source waters ...... 17

1.6.1 Sampling for Norovirus and Enterovirus from source waters ...... 18

1.6.2 Concentration of Norovirus and Enterovirus from source waters ...... 19

1.7 Detection and Enumeration of Norovirus and Enterovirus from source waters ...... 23

1.7.1 Cell culture ...... 23

1.7.2 Polymerase Chain Reaction (PCR) ...... 24

1.7.3 Immunoassay or enzyme linked immunosorbent assay (ELISA) ...... 26

1.7.4 Integrated Cell Culture (ICC) PCR ...... 26

1.8 Qualitative Microbial Risk Assessment (QMRA) ...... 27

Chapter 2: Materials and Methods ...... 32

2.1 Norovirus and Enterovirus Nucleic Material ...... 32

2.2 Source Water Sampling...... 32

2.2.1 Water Sample Site Locations and Collection Technique ...... 32

2.2.2 Ground Water (GW) Site Description ...... 32

2.2.3 River Water Site Description ...... 33

2.2.4 Wastewater (WW) ...... 33

2.3 Sampling Plan ...... 34

2.3.1 River conditions and water quality observations ...... 35

2.3.2 Sampling Technique ...... 36

2.4 Concentration of water samples ...... 38

2.5 Nucleic acid extraction ...... 41

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2.6 Reverse Transcription and Quantitative Polymerase Chain Reaction (RT-qPCR) ...... 42

2.7 Preparation of Plasmid Standards (Standard curve) ...... 42

2.8 Method Performance Spike Evaluation with MS2 Bacteriophage ...... 43

2.9 Determination of qPCR Inhibition using Standard Plasmids ...... 44

2.10 Quantitative PCR Assay for Norovirus and Enterovirus ...... 46

2.11 Statistical evaluation ...... 47

Chapter 3: Results ...... 49

3.1 MS2 recovery ...... 49

3.2 PCR Inhibition by Environmental Samples ...... 51

3.3 Quantification of Norovirus and Enterovirus by qPCR ...... 53

3.4 Environmental trends: ...... 59

3.5 Community prevalence and outbreak surveillance: ...... 63

3.6 Statistical correlations: ...... 64

Chapter 4: Discussion ...... 68

4.1 Recovery of MS2 as a surrogate using USEPA 1615 ...... 69

4.2 Prevalence of Norovirus and Enterovirus ...... 70

4.3 Seasonal Occurrence of Norovirus and Enterovirus ...... 71

4.4 Meteorological, Physical and Chemical Interactions ...... 73

4.5 PCR detection and determination of infectivity of Norovirus and Enterovirus ...... 75

4.6 Bacterial Indicators as surrogates for Norovirus and Enterovirus ...... 76

5.0 Future Perspectives ...... 78

REFERENCES ...... 80

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APPENDICES ...... 93

Appendix 1: Sampling Record ...... 94

Appendix 2: Tables of study data ...... 96

Appendix 3: Inhibition test results ...... 99

Appendix 4: Formulations ...... 118

Appendix 5: Calculations...... 120

Appendix 6: Jurisdictional scan of water quality standards ...... 123

List of Tables

Table 1.1 Disease spectrums of human Enterovirus types

Table 1.2 Norovirus and Enterovirus concentrations found across water matrices from selected publications

Table 1.3 Filter options available to concentrate enteric viruses from waters

Table 2.1 Sample collection between December 2014 and April 2016

Table 2.2 PCR Master Mix for Norovirus and Enterovirus assay and PCR conditions

Table 2.3 Primers and TaqMan® Probes for Virus Detection by RT-qPCR

Table 3.1 Samples positive for human Norovirus and human Enterovirus as determined by qPCR

Table 3.2 Wastewater treatment grab samples positive for human Norovirus GII and Human Enterovirus types as determined by qPCR

Table 3.3 Statistical calculation of correlation between virus count (GC/L) in River Water and physical variables

Table 3.4 Statistical calculation of correlation between virus count (GC/L) in Ground Water and physical variables

List of Figures

Figure 1.1 The sources and potential reservoirs for human Norovirus and human Enterovirus originating from a human faecal source.

Figure 1.2 Diagram of reported Echovirus (Enterovirus B) Infections in Canada from 2005 to 2009.

Figure 2.1 The virus sampling apparatus at the raw river water intake sampling line

Figure 2.2 A schematic diagram showing the stepwise approach used in this study from field sample collection to qPCR assay

Figure 2.3 The laboratory sample elution apparatus and set-up

Figures 3.1 Human Norovirus GII and human Enterovirus counts for untreated (raw) ground water source

Figures 3.2 Human Norovirus GII and human Enterovirus counts for untreated (raw) river water source

Figures 3.3 Time series plot for January 2015 through to April 2016 of human Norovirus GII and human Enterovirus counts vs. Precipitation for untreated (raw) ground water source

Figures 3.4 Time series plot for January 2015 through to April 2016 of human Norovirus GII and human Enterovirus counts vs. Precipitation for untreated (raw) river water source

Figure 3.5 Time series plot for January 2015 through to April 2016 of river flows and counts of human Norovirus GII and human Enterovirus.

Figure 3.6 Plot of Norovirus count vs. Temperature data for January 2015 through to April 2016

List of Appendices

Appendix 1 Table of sampling plan

Appendix 2 Table of study data for all processed samples from river water and ground water

Appendix 3 Table of inhibition study data for all processed samples from river water and groundwater

Appendix 4 Table of formulations used for Inhibition and PCR assays

Appendix 5 Calculation used to determine the genome copies per liter of raw water

Appendix 6 Table of international jurisdictions drinking water and source water microbiological guidelines

and standards

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List of Abbreviations BE Beef extract BGS Below ground surface bp Base pair CDC Center for Disease Control cDNA Complementary deoxyribonucleic acid CI Confidence interval CPE Cytopathic effect CT Concentration times contact time Ct Cycle threshold DI Distilled DNA Deoxyribonucleic acid DWTP Drinking water treatment plant E. coli Escherichia coli EMA Ethidium monoazide EV: Human Enterovirus types FAM Fluorescein. A fluorescent dye attachment for oglionucleotides FCV Feline Calicivirus g Gram GC/L Genome copies per litre GRCA Grand River Conservation Authority GUDI Ground water under the influence of surface water GW Ground Water (untreated raw) h Hours HCl Hydrochloric acid IEP Isoelectric point. This is pH value at which the electrostatic surface charge on the viral particle, in water, is neutral IPTG Isopropyl β–D-1-thiogalactopyranoside is a compound that mimics a lactose metabolite to trigger transcription of the lac operon Kb Kilo base Km Kilometer L Litre LB Luria-Bertani agar L/min Liters per minute LOD Limits of Detection (no amplification seen during qPCR) LOQ Limits of Quantification (≥3 GC per PCR reaction) M Molar

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mg Milligram MIQE Minimum information for publication of quantitative real-time PCR experiments mL Millilitre mm Millimeter mM Millimolar MNV-1 Murine Norovirus MOECC Ontario Ministry of the Environment and Climate Change MS2 Bacteriophage of E. Coli N Normality NaOH Sodium hydroxide ng Nanograms nM Nanomolar NoV GII Human Norovirus genogroup II NSP Non-structural protein of Norovirus genome ORF1 Open reading frame number 1 ORF 2 Open reading frame number 2 ORF 3 Open reading frame number 3 P Probability (≤0.05 has been assigned for significance) PCR Polymerase chain reaction PFU Plaque forming units PHAC Public Health Agency of Canada PMA Propidium monoazide ppm Parts per million QMRA Quantitative microbial risk assessment qPCR Quantitative polymerase chain reaction r Pearson Correlation Coefficient (strength of linear association) RNA Ribonucleic acid rpm Rotations per minute RT-qPCR Reverse transcription quantitative polymerase chain reaction RW River Water (raw untreated) s Seconds TAMRA Quencer TE Tris-EDTA (buffer) U Units µg Microgram µL Microlitre

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USA United States of America USEPA United States Environmental Protection Agency UTR Untranslated region. Also known as a leader sequence (5’UTR); or the section immediately following translation termination codon (3’ UTR) UV Ultraviolet VIRADEL Virus adsorption and elution VP1 Virus protein number one of Norovirus genome VP2 Virus protein number two of Norovirus genome VPg multi-functional protein essential for virus replication WHO World Health Organization WW Wastewater WWTF Wastewater treatment facility X-gal Also known as BCIG (bromo-4-chloro-3-indolyl-β-D-galactopyranoside) to test for the activity of the enzyme β- galactosidase

Chapter 1: Introduction and Literature Review

1.1 Waterborne disease from human enteric viruses

Human enteric pathogens, including enteric viruses, are significant global causes of disease and the consumption of contaminated water is a well-documented vehicle for the transmission of gastroenteritis disease (WHO, 2004). Impacts of waterborne diseases occur in both developed and underdeveloped nations, with the latter notably reporting higher disease rates

(Nicole, 2015). Entry of human enteric pathogens into water can follow multiple pathways including wastewater treatment effluents, septic tanks, or run-off from land application of manures or biosolids (Figure 1.1). In addition to direct consumption of contaminated water, transmission of enteric viruses can occur via person-to-person contact, contaminated foods, and fomite contaminated surfaces once released into the environment from the faecal origin (Figure

1.1). Two broad groups of enteric viruses that are significant contributors to waterborne illnesses are the human Norovirus and Enterovirus groups.

Enteric viruses, such as human Norovirus and human Enterovirus, are highly contagious, have a low infectious dose and infect persons of all ages (Rockx et al. 2002). Human Noroviruses are now the most common cause of gastroenteritis outbreaks in children in developed countries worldwide and principal etiological agents implicated in food borne disease outbreaks in the

United States of America (USA) (Lopman, 2016). Although countries such as the USA and

Canada have realized reductions in waterborne disease outbreaks attributable to the effectiveness of water quality regulatory frameworks and treatment technologies (IOM, 2009), illness and outbreaks continue to be reported each year. Health care facilities such as hospitals and nursing homes; schools; cruise ships and catered events are most commonly reported settings for Norovirus outbreaks in developed countries. Enterovirus, one of the largest genera

of viruses classified within the Picornaviridae family, represents a significant burden to public health globally and was estimated in 1986 to cause an estimated fifteen million infections each year in the USA alone (Strikas et al. 1986). Thus, the monitoring of these human pathogens can play a critical role in maintaining safe water supplies.

The purpose of this review is to outline current knowledge of human Norovirus and Enterovirus as agents of waterborne disease with an emphasis to their presence in source waters to be used for drinking. Relevant information gaps of the prevalence and persistence of human

Noroviruses and Enteroviruses in surface and ground waters will be identified. The impacts of seasonality will be described with respect to regions with temperate climates, as this can play an important role in the presence of these viruses in source waters. The current methods used for the concentration, detection and enumeration of these viruses will be summarized, as the selection of appropriate methods can impact the ability to detect these viruses from different environmental matrices. Lastly, the role of each of these areas will be described within the context of quantitative microbial risk assessment, which is an important tool in maintaining safe drinking water supplies.

Septic systems Combined sewer overflows

Soil and or crops

Wastewater treatment

facility Effluent

Industrial treatment facility Sludge

Water treatment facility

Source Water

Figure 1.1 The sources and potential reservoirs for human Norovirus and human Enterovirus originating from a human faecal source.

1.2 Norovirus

1.2.1 Agent

In 1972, Norovirus was first discovered using immuno-electron microscopy as the causative agent of a gastroenteritis outbreak in 1968 in an elementary school in Norwalk Ohio. Norwalk viruses also known in the literature as, Norwalk like viruses (NLV), Norovirus (NOVs), human

Norovirus (HuNOVs) and previously referenced as small round structured viruses (SRSVs,

SRVs) are classified within the Caliciviridae family. Caliciviruses are icosahedral capsid shaped small (~38nm in diameter) non-enveloped, single-stranded, positive-sense RNA viruses containing a genome of 7.5 to 7.7Kb in length (Bull et al. 2005). Additional names for Norwalk virus types that appear in the literature such as Hawaii, Snow Mountain and Taunton were derived from the site of the outbreak where human Norovirus was identified as the causative agent.

Human Norovirus infection is characterized by a short incubation period of one to two days and presents symptoms of stomach upset, vomiting and diarrhea that resolves within one to three days (CDC.gov/Norovirus/ last accessed July 2016). The infectious dose of Norovirus has been estimated to be as low as 18 virions during human subject (subject’s age, race and health status not disclosed) feed trials (Teunis et al. 2008). Norovirus has been reported to shed in the stool of human subjects for up to two (2) weeks after infection (CDC, 2011). Norovirus has been reported to shed in infected stool at concentrations of up to 100 billion viral genomic copies per gram of faeces and are capable of persisting in the environment and as such are transmissible to drinking water via sewage-contaminated source waters (surface and ground waters) (CDC,

2011).

1.2.2 Taxonomy

The International Committee on Taxonomy of Viruses, as of its last approval in March 2016, has currently recognised five (5) genera within the Caliciviridae Family, comprised of Lagovirus,

Nebovirus, Vesivirus, Sapovirus and Norovirus. Viruses within the Norovirus and Sapovirus genera are known to cause disease specifically in humans (http://www.ictvonline.org/, last accessed July 9, 2016). Complete genome sequencing for human Caliciviruses is available from the Genbank database. Presently 51 human Norovirus genomes have been sequenced

(https://www.ncbi.nlm.nih.gov/nuccore, last accessed July 2017). Partial genome sequencing from the polymerase and capsid protein genes are also available and have been reported to be used for phylogenetic analysis resulting in the creation of genogroups (Etherington et al. 2006).

For instance, Zheng et al. (2006) performed a capsid sequence study in 2006 that resulted in the further dividing of Norovirus into five groups (GI, GII, GII, GIV and GV) of which GI, GII and

GIV contain human isolated Noroviruses, while GIII represents bovine and GV murine

Noroviruses. Presently, Norovirus GII is the most prevalent type causing gastroenteritis illness worldwide (Glass et al. 2009). Animal strains of Norovirus are not known to infect humans.

1.2.3 Structure of the genome

The Norovirus genome contains three open reading frames (ORF1, ORF2, and ORF3) that encode structural and non-structural proteins. The genome is known to have eight major structural proteins. VP1 is a major structural capsid protein within ORF2 that has been reported to self-assemble into the viral particles icosahedral shape as verified through electron microscopy (Donaldson et al. 2010; and Jiang et al. 1992). This major structural protein is thought to possess a stable conserved protein sequence since it determines the morphology of

Norovirus. As such the VP1 gene sequence has been used as a genetic marker to detect and identify human Norovirus (Fankhauser et al. 2002; and Vinje et al. 2015).

1.3 Enteroviruses

1.3.1 Agent

Viruses of enteric origin were discovered during investigations into the spread of poliomyelitis.

Paul and Trask’s work in the 1940’s identified Poliovirus in faecal material, spawning development of methods to investigate viruses of enteric origin (Paul and Trask, 1942).

Presently there are over 200 viruses that reside within the Picornaviridae Family. Viruses within this family range from 20 to 30 mm in diameter are icosahedral shaped, non-enveloped, single- stranded, positive-sense RNA viruses containing a genome of 7.2-8.5 Kb (Hellen and Wimmer,

1995). The genome is composed of a single ORF encoding a polyprotein which is subsequently processed into capsid proteins via proteases. The genomic RNA has a viral protein at its 5’end and a 3’ polyA tail. The long UTR at the 5’ end contains an internal ribosome entry site.

Enterovirus has four (VP1, VP2, VP3 AND VP4) capsid proteins, of which the VP1 gene is considered a stable structural protein in the assembly of the icosahedral shape (Hellen and

Wimmer, 1995). This large group of viruses target either intestinal or upper respiratory tract cells resulting in an upper respiratory tract infection or gastrointestinal illness. Enterovirus types can cause a wide spectrum of diseases within humans and present a broad range of symptoms

(Table 1.1). Enterovirus infectious dose, incubation periods, and symptoms of Enterovirus illness depend on the virus species, and, similar to Norovirus are also transmissible via sewage contaminated waters (Health Canada, 2012).

Table 1.1 Human Enterovirus diseases Genus Common name Disease caused (not all inclusive) Enterovirus Coxsackievirus A,B Gastroenteritis, Hand-foot and mouth, respiratory disease, fever, heart anomalies, meningitis, Guillian-barre syndrome, Echovirus Gastroenteritis, fever, respiratory disease, meningitis

Poliovirus Paralysis, meningitis, fever

Adapted from http://www.enterovirusfoundation.org/associations.shtml

1.3.2 Taxonomy

The Enteroviruses were originally classified into four groups: Polioviruses, Coxsackievirus A,

Coxsackievirus B, and Echoviruses. This was based on the physical structure of the viruses, the tissue cultures in which they would grow, and their pathogenesis in humans and experimental animals. Today, they are classified based on the virus genomic structure. The

International Committee on Taxonomy of Viruses recognises thirty one (31) genera within the

Picornaviridae Family that currently contains 54 species (http://www.ictvonline.org/ Last accessed 28 October 2016). The Enterovirus genus is comprised of Enterovirus species

A,B,C,D,E,F,G,H, J ; and Rhinovirus A,B,C. Presently, only viruses within the Enterovirus A, B,

C, D and J are known to cause disease in humans. Subtypes included in this Genus include:

Echovirus (Enterovirus B); Coxsackieviruses (Enterovirus groups A, B and C). According to

PHAC (2015), Echovirus 30 is the most common non-polio Enterovirus circulating in Canada based on outbreak data since 1998.

1.4 Seasonality of Norovirus and Enterovirus

Norovirus outbreaks can occur throughout the year, but have been reported to occur more frequently during the colder winter seasons between November and April in temperate climates

(CDC, 2014). Maunula et al. (2005) investigated 18 waterborne outbreaks in Finland between

1998 -2003 and concurred that the majority of outbreaks of Norovirus occurred late winter to early spring. Further, Ahmed et al. (2013) conducted a systematic review of publications between 1998 and 2001 and reported Norovirus cases and outbreaks exhibited seasonality with a peak in winter months and a trough in summer months. This seasonality was associated with average rainfall of the wettest month for both the Northern and Southern Hemispheres within

Europe and Asia. They also reported a lack of data in the Americas, Africa and Australia to summarize seasonal strength. Australia, (OZFoodnet, 2011) reported Norovirus outbreaks most commonly from food and person to person transmission in the months of May to September, which correspond to that region’s late fall and winter season. Schuster et al. (2005) also reported seasonal patterns for waterborne outbreaks in Canada and stated twenty five out of

223 of known factors contributing to waterborne outbreaks (10%) were attributable to meteorological weather events. Norovirus was identified as the causative agent in 14 outbreaks between 1974 and 2001 out of 150 outbreaks where an etiological agent was identified.

Further, Greer et al. (2009) performed a retroactive review and reported a potential correlation between Norovirus outbreaks and lake water temperatures in winter, inferring lake water used for drinking sources are a reservoir for enteric viruses in these environmental matrices and conditions. However, no environmental monitoring and surveillance programs were in place at the time to confirm this association. The above studies informed this investigations approach to sampling across multiple seasons with targeted sampling following precipitation events.

Human Enterovirus outbreaks can occur throughout the year depending on the type of

Enterovirus. In temperate climates, Enterovirus infections are most prevalent during summer months. Prominent summer-fall seasonality in the USA has been reported for non-polio

Enteroviruses (Moore, 1982); with a peak annual incidence occurring in August for Echovirus and Coxsackievirus A and B viral infections, based on Enterovirus surveillance data from the

United States between 1970 and 1979. Sedmak et al. (2003) identified Enterovirus infection rates corresponding to late summer in the USA, with Coxsackievirus B1 as the most common

(21%) followed by Echovirus 3 and 7 (19% and 34% respectively), whereas Poliovirus was not detected. Costan-Longares et al. (2008) reported cases corresponding to early summer in

Spain with Coxsackievirus B4 as the most commonly isolated serotype (70-100%), followed by

Echovirus (23-27%) and Poliovirus (0-3%) from wastewater, river water and seawater. In

Canada, through genotyping of specimens from select cases, Echovirus has been identified as one of the leading Enterovirus types causing illness (Figure 1.2). Positive tests analyzed at the

Public Health Agency of Canada (PHAC) illustrate Enterovirus infections peak in the late summer and early autumn months (PHAC, 2015).

Figure 1.2 Reported Echovirus (Enterovirus B) Infections in Canada from 2005 to 2009. Seasonality is observed through the increased number of positive detections conducted by PHAC laboratories, reported during summer months June to September. Graph adapted from data on the Public Health Agency of Canada website (PHAC, 2015)

Both Norovirus and Enterovirus have exhibited seasonality in outbreak occurrences, which can be impacted by the effects of global climate change. Understanding the relationship between climatic factors and enteric viruses can be useful at the local watershed level where quantitative microbial risk assessment models rely on multiple parameter inputs. Further, global climate change can potentially alter the distribution and occurrence of waterborne gastroenteritis disease. In Canada, precipitation and temperature are predicted to increase according to the Department of Natural Resources Canada

(http://www.nrcan.gc.ca/environment/resources/publications/impacts- adaptation/reports/assessments/2008/ch2/10321, last accessed August 2017), and this may alter the prevalence and occurrence of Norovirus and Enterovirus in Ontario and Canada.

1.5 Prevalence and persistence of human Norovirus and human Enterovirus in source waters

Host exposure and infection depend on the persistence, survival and transport of the viruses in the environment. In addition to surviving in the gut long enough to initiate infection (gastroenteritis) within a susceptible host; enteric viruses must survive transport through various environmental pathways once released in the faecal waste from an infected individual to reach the next potential host. Sources and reservoirs of human enteric viruses are shown in Figure 1.1. As Norovirus and Enterovirus are primarily transmitted through the faecal to oral route, wastewater effluents, and leaching septic tanks are a primary source and reservoir of these viruses released into receiving waters, which in turn potentially contaminate source waters for drinking (Okoh et al. 2010). Wastewater treatment facilities may release viruses to environmental waters through untreated (by-pass events or combined storm-sewer) or treated effluent discharge. Both human Norovirus and human Enterovirus have been detected in wastewater effluents that have been treated or disinfected (Symonds et al. 2009). Qiu et al. (2015) reported 6% of final effluents treated by ultraviolet radiation contained human Norovirus and human Enterovirus. Simmons et al. (2011) reported wastewater to contain enteric viruses in concentrations ranging between 103 and 109 viral particles

per liter. In this study, thirty wastewater samples over five months were sampled and analyzed for the presence of human Adenovirus, Enterovirus and Norovirus genogroup II. Using real-time PCR and cytopathic effect, viruses were detected in all samples at the following concentrations: for influent (raw wastewater) 4.1 MPN/100L; pre and post disinfection of 1.1 and 0.5 MPN/100mL respectively. Additional published data on the concentration of Norovirus and Enterovirus in wastewater are summarized in Table

1.2.

The ability of enteric viruses to survive in natural and man-made environments involved in the treatment of drinking water is dependent upon the multifaceted interactions that affect the viral particles within the water matrix. Viral particles in water behave as biocolloids (Tchobanoglous and Schroeder, 1985) and as such, natural organic materials, pH, and ionic strength of water matrices can all affect transport and attachment or dissociation of the viral particles. In particular are the interactions to surfaces such as organic material and sediments within the natural environment, and filter media within water treatment processes (coagulants, filter sands). Survival of Norovirus and Enterovirus in the natural environment also depends on physical and biological variables such as temperature, exposure to ultraviolet (UV) light, and presence of microbiological flora (Bosch et al. 2006). These are discussed in relation to Norovirus and Enterovirus in further detail below.

The occurrence of human enteric viruses in surface water and ground waters has been investigated in many countries. River water samples in France (Prevost et al. 2016) were positive for at least one target virus at median viral loads ranging from 10 to 50 genomic copies (GC) /L for Enteroviruses and Noroviruses by real time quantitative polymerase chain reaction (RT-qPCR). Lazic et al. (2015) reported Norovirus GII and GI were detected in over 40% of river waters tested in Serbia. Marcheggiani et al. (2015) reported 103 (GC)/L for both Norovirus types GI and GII within the Mezzocammino river in Italy; and no detections for

Enterovirus from river water in Italy. Occurrences of enteric viruses, such as human Norovirus GII (2% of samples) and human Enterovirus (20% of samples) were detected in concentrations of up to 8.74x104 and

9.29x102 GC/L, respectively, over the course of a twelve month study of fresh water lakes in Michigan

(Aslan et al. 2011). In Canada, Payment et al. (1988) reported human enteric viruses (types not specified)

were detected in 13 to 72% of water samples from the Assomption River and 14 to 71 % of water samples from tributaries, sampled between June and November over two years. In addition, seasonal patterns were observed for all viruses with an increase in viral concentration in early fall. Wong et al. (2009) also reported

3-12% of samples collected between June and September 2004, at two sites within Lake Michigan were positive for human Enterovirus. Enteric Viruses have also been detected in sediments below the water column. Ferguson et al. (1996) suspected that sediments can act as reservoirs for enteric viruses. In their investigation, they took samples from an urban estuary and detected viruses primarily in water and top sediment, whereas no viruses were found in the bottom sediment. Resuspension of enteric viruses back into the water column could pose a risk to human health (De Flora et al. 1975; and Malham et al. 2014).

Enteroviruses have been shown to adsorb to solid matter within a water matrix, and therefore may travel long distances in water or settle out of the water to become concentrated in sediments below the water column (Malham et al. 2014).

Human enteric viruses have been detected in ground water. The U.S. Geological Survey reported about 8% of wells positive for culturable human enteric viruses (Davis and Witt 1998). In the USA, a nationwide study, sampled 448 groundwater sites in 35 states for the presence of Enterovirus, Rotavirus, Hepatitis A virus, and Norovirus. Viral nucleic acid from at least one of these four viruses was present in 31% of sampling sites (Abbaszadegan et al. 2003). Fout et al. (2003) sampled 29 groundwater sites and reported the presence of human enteric viruses, primarily Reovirus and Enterovirus, in 72% of 29 groundwater sites within the USA sampled over a one-year period. Borchardt et al. (2004) sampled drinking water wells in

Wisconsin monthly for a period of a year and detected Echovirus and Coxsackievirus group A&B by RT-

PCR in 42% of the samples and Norovirus GI in 6%. In Canada, a study of municipal wells in the province of Quebec found enteric viruses in 4 of the 12 sites and Norovirus in 3 of the 12 sites over a one-year period, however sampling dates were not included and no statement on seasonal correlations was made

(Locas et al. 2007). Viruses and other microorganisms can survive for several months in soil and ground water when temperatures are lower (4-12° C) and soils are moist (Yates et al. 1985). Further, microbial transport can be facilitated in saturated soil (Jamieson et al. 2002) or by preferential flow (Shipitalo and

Gibbs 2000). Viruses have been reported to migrate 1,600 m in fractured limestone (Keswick and Gerba

1980). In groundwater, the presence of indigenous microorganisms (not identified) is an important feature in the decay of Enterovirus (Gordon and Toze 2003).

Table 1.2 Norovirus and Enterovirus concentrations found across water matrices Matrix Virus type Concentration Reference Comment

Wastewater influent Norovirus GII 1.8 to 5.7 log10 GC/100 Carter (2005); Concentrations are and GI mL; 3691 GC/mL; 104 Zheng et al. (2006); dependent on enteric PFU/L virus type surveyed (GII concentrations were reported greater than GI)

Wastewater effluent Norovirus GI & 2.53 and 2.63 log10 GC/ Flannery et al. 1 L grab sample GII 100 mL−1 (2012) secondary treated wastewater; also

reported increase concentration in winter (P<0.05), Ireland

0.5 to 4.3 log10 GC/100 Zheng et al. (2006) mL

River water Enterovirus 1 to 6 PFU/L Lodder and Belgium rivers Husman, 2005) influenced by un- Norovirus 2x102GC/L treated sewage from

France and Belgium.

Dorner et al. (2007), Enteric viruses 0-6.4 MPN/L of river Grand River, Ontario water Canada

Ground water Enterovirus 0.8 GC/L (mean) Bouchard et al. Treated groundwater (2012) sampled in homes at Norovirus 0 to 0.6 GC/L the tap, USA

Enterovirus Not detected to 19.2 Treated and untreated GC/L Lambertini et al. groundwater, USA. (2008)

1.5.1 Survival and Inactivation of Norovirus

There is little research available on the survival and persistence of human Norovirus in the environment.

Many studies have relied on spiking samples with high concentrations of a surrogate virus and following their survival over time under laboratory conditions, which may not be representative of conditions in the field. Detecting Norovirus in environmental samples by their nucleic acid has the limitation of not being able to conclude if the detected viruses are infectious. Viruses, infectious or inactivated are both detectable by

PCR assay. Traditional survival studies of viruses require propagation of infectious virus in cultured cells, although this has recently been demonstrated for in vitro cultivation of human Norovirus in highly differentiated intestinal epithelial cells and within human B cells (Jones et al. 2015; Papafragkou et al. 2013) there is no validated standardized methodology for use with environmental samples. Alternatively, surrogates such as murine Norovirus (MNV-1) and feline Calicivirus (FCV) can be propagated in vitro cell culture, and have been relied on previously to make inferences about the survival of human Norovirus because of structural similarities (Papafragkou et al. 2013).

Norovirus has been reported to be stable when exposed to and chlorine disinfection treatments (200 ppm chlorine solution) (Predmore and Li, 2011). Human Norovirus was reported to remain infectious after exposure to pH 2.7 for 3 hours and after heating at 60°C for 30 minutes as demonstrated in human volunteer studies (Dolin et al. 1972). Flynn et al. (1988) reported that Norovirus – like –particles (NV VLP) have less stability when exposed to pH 3-7 up to 55°C. Differing conclusions may suggest that the virus type investigated or medium composition could favourably influence virus survival. Allwood et al. (2003) reported reductions in infectivity of both MS2 and FCV (surrogates for Norovirus) when storage temperatures were increased from 4° to 37° C

Human Norovirus has been reported to remain infectious after contact with chlorine at concentrations of up to 10 mg/L (ppm), as shown in a study where human volunteers were fed Norovirus treated with 10mg/L of chlorine for 30 minutes (Keswick, 1985). The United States Environmental Protection Agency recommends a residual of 0.1 mg/L free chlorine in its drinking water distribution systems; this suggests that Norovirus

may survive disinfection treatment processes if infectious viral particles enter the distribution system. In

Ontario Canada, a chlorine disinfection residual in the drinking water distribution system of 0.25 to 0.05 mg/L (monochloramine and chlorine/chlorine dioxide, respectively) is required for systems primarily disinfecting with chlorine (Ontario Regulation 170/03), thus a similar situation to the US could occur. In

Ontario, primary drinking water treatment, which includes technologies such as coagulation and filtration, is required to achieve 99.9% removal of viral pathogens before entering the distribution system (Ontario

Regulation 170/03). Feline Calicivirus (FCVs) have been studied as a surrogate for human Norovirus.

Seah et al. (1999) reported FCV inactivation by >4 log within 5 min with a dose of 30 mg/L chlorine, therefore conventional water treatment facilities would require mixing and contact times of the water with the chlorine for a minimum of 5 minutes to achieve the 4 log reduction in Norovirus before water was released into the distribution system. FCV has been well studied and used as a surrogate for human Norovirus, however FCV is transmitted through the respiratory route, and may not be truly representative of human

Norovirus which is transmitted via the fecal to oral route. As such, Cannon et al. (2006) compared the stability and inactivation of murine Norovirus (MNV-1), transmitted via the fecal oral route in mice, to FCV to see if inactivation results differed between the two virus types and determine their relevance as potential surrogates for human Norovirus. They reported MNV-1 was more stable at ambient temperatures and more stable at lower pH environments than FCV and suggested that MNV-1 was a preferred surrogate for studying environmental survival of human Norovirus than FCV.

1.5.2 Survival and Inactivation of Enteroviruses

Cosackieviruses have been reported to remain viable at temperatures between -20°C and -70°C for years and for weeks at 4°C, while they tend to lose infectivity as temperatures increase (WHO, 2005). In seawater at 15°C, Poliovirus was observed to survive for many days, a reduction of up to 3 logs was observed after

28 days (Enriquez et al. 1995). In freshwater, human Enterovirus were reported to survive for several weeks. Coxsackievirus B3, Echovirus 7, and Poliovirus 1 were inactivated by 6.5–7 logs over 8 weeks at

22°C, and 4–5 logs over 12 weeks at 1°C (Hurst et al. 1989). There are many publications on the effects of disinfectants as well as UV light on Enteroviruses. For instance, It has been reported that exposure to UV

light or sunlight can enhance virus inactivation in the environment. Cook and Rzezutka (2004) reported an inactivation rate of 99% for poliovirus in the absence of UV light in marine water after 52 days, whereas in the presence of sunlight, only 21 days were required. UV irradiation can penetrate the viral particle capsid, damage genetic material, and therefore interfere with replication. Disinfection with UV light may depend on

UV lamp type. For instance, medium-pressure UV lamps can achieve higher inactivation rates compared to low pressure lamps at the same total intensity (Linden et al. 2007). Higher UV dose can steadily increase inactivation of a variety of viruses such as Echovirus, Coxsackievirus, and Poliovirus (Gerba et al. 2002a).

Shin and Sobsey (2008) reported that inactivation of Poliovirus and a solution of purified and dispersed

Norovirus was enhanced with higher dose of chlorine (5 mg/L), even though the contact time was shorter

(10 min). Thurston-Enriquez et al. (2003a) showed that the virus removal (Poliovirus 1) was directly related to contact time. Ozone is more effective than chlorine for virus disinfection, but similar to chlorination, in that higher doses of ozone and longer contact time generally result in better performance for virus inactivation.

Katzenelson et al. (1979) reported the log removal of Poliovirus doubled when the ozone dose increased from 0.4 to 1.24 mg/L. More recently, research has ensued on Enteroviruses of respiratory public health significance such as Enterovirus 68 and 70.

1.6 Methods for the Collection and Detection of Norovirus and Enterovirus from source waters

Detection of human enteric viruses from environmental matrices is considered challenging because of the variability of illness and shedding of viral particles within any given community at any given time; the natural chemical, physical, biological and hydrological properties of the environment; presence of components that may interfere with detection of viral particles; and the heterogeneous distribution of viruses in those environments. Environmental waters (ground water, rivers, lakes, and sea waters) exhibit seasonal, temporal and geographic variability, adding another layer of complexity for consideration when monitoring for viral particles.

The ability to detect the viral particle (agent identification) and determine its infectivity is used to predict risk of infection, yet not all enteric virus types can be cultivated in-vitro (Lee and Jeong 2004). Active research continues in the development of protocols to address limitations of the detection of enteric viruses in environmental waters. Limitations include; dilution of the viral particles once the viral particles enters source waters; and the effect of environmental physical factors such as temperature, and UV light.

Consequently, methods must be sensitive, capable of discerning virus presence in the face of matrix interferences, and specific to detect the enteric virus of interest. To counteract the effects of dilution, several viral concentration methods have been developed, which are described in detail in Section 1.6.2 below. A common approach employed in the field collection and detection of enteric viruses in waters is the three-step approach consisting of: sample concentration of large volumes of raw water; elution; and re- concentration prior to viral assay.

1.6.1 Sampling for Norovirus and Enterovirus from source waters

Viral particles become diluted once they are shed from an infected individual, enter wastewater collection and treatment systems, and become further diluted once remaining surviving virions in treated effluents are discharged into a receiving body of water (sea, lake, river, stream or soil). Information on dilution effect within wastewater collection systems; and dilution effect on wastewater effluent discharging into receiving source waters have not been well described in the literature (Payment et al. 2001). To address dilution of enteric viruses in the environment, sampling of large volumes of water is required. Collecting and transporting large volumes of water, hundreds to thousands of litres, is not practical, and as such on-site filtration is the method of choice when sampling for enteric viruses such as Norovirus and Enterovirus from source waters. Sampling strategies are dependent on the water type and amount of contamination (sewage pollution). The USEPA method 1615, adopted by a number of researchers interested in the determination of enteric viruses from environmental water (Cashdollar et al. 2013; and Mclellan et al. 2016) recommends sampling between 120 L for wastewater effluents, 360 L for surface waters, and between 1800 L to 4,320 L for groundwater. Additional challenges associated with sampling of large volumes include the simultaneous concentration of compounds that may interfere with downstream elution, re-concentration, or nucleic acid

extraction steps. For example, humic acids, natural organic materials and other organisms that adsorb to the surface of the filter medium. Table 1.2 summarizes the concentrations of Norovirus and Enterovirus found across water matrices from selected publications. From the studies reviewed, it was observed that ground water samples reported the lowest concentration of virus present and the range of virus concentrations reported from wastewater influent and effluent samples was similar to those reported for surface water sources

1.6.2 Concentration of Norovirus and Enterovirus from source waters

Norovirus and Enterovirus are present in relatively low numbers in source waters, and therefore require concentration prior to quantification. Although no single method exists for the recovery and detection from water, Standard Methods; (APHA, 2011) identifies three different techniques for concentrating viruses: absorption to and elution from microporous filters; adsorption – precipitation using aluminum hydroxide and hydroextraction – dialysis using polyethylene glycol (PEG). The goal of concentrating is to collect the greatest number of viral particles in the smallest volume possible in the event low numbers of viral particles are circulating in the source water at the time of collection.

Since the 1930s, when Schlesinger discovered that viruses could adsorb to colloidal membranes, active research into the development of a filter to permit the filtering of large volumes of water in a short time without clogging or concurrently concentrating inhibitory substances has been pursued. Briefly, it is necessary to concentrate viruses from environmental matrices before performing tests for detection, selecting the concentration approach must consider the virus type and should not be cytotoxic to cell cultures used in infectivity assays and should be free of any inhibitors, which may be co-extracted or co- concentrated from environmental samples. Filter materials include nitrocellulose, epoxy fibreglass, glass and electronegative membrane materials. The filter selection depends on water type (sewage effluent, groundwater) and which virus type is of interest as enteric viruses carry different surface charges and behave differently (attach to the filter media or elute from the filter) at pH that corresponds to their respective surface isoelectric points. As the majority of enteric viruses have been found to be negatively charged at pH

2-7 (Michen and Graule, 2010), positively charged filters are commonly used. Positively charged filters do

not require a water conditioning step to facilitate binding; allow large volumes of water to be filtered (without the addition of salts) and are more economical than negatively charged filters (Karim et al. 2009).

Knowledge of isoelectric points (IEP) and source water pH can inform choice of filter. The IEP for human

Norovirus and FCV was determined o be close to pH 4. The strongest negative surface charges were reported at pH 6 whereas the lowest negative charge was recorded at pH 3 (Jean et al. 2015). (Michen and

Graule 2010) investigated the electrofocusing of Enterovirus B and C, Rhinovirus and Poliovirus. Poliovirus

1 was found to have an isoelectric point of 6.6; Rhinovirus B and C were 6.4 and 6.8, respectively; and human Echovirus was reported to be between 4.0 and 6.4; human Coxsackievirus B was reported at 4.75 and 6.75. Most natural waters in Northern America have an average pH level of 6.5 or 8.5, for example the pH of river water in Ontario’s Grand River is between pH 8 and 9 as monitored and measured by the Grand

River Conservation Authority (GRCA, 2016 https://apps.grandriver.ca/waterdata/kiwischarts/wq_ph.aspx), thus concentrating Norovirus and Enterovirus from source waters by adsorption would support the employment of a positively charged filter medium, which is the approach used by the United States

Environmental Protection Agency (USEPA) as per the USEP 1615 protocol and the method used in this study.

Once collected by filtration, an elution procedure is required to remove the virus particles from the filter.

Different approaches to filtering and eluting have been pursued. Table 1.3 presents some of the options available to concentrate viruses from environmental waters. No one single filter type and elution method recovers all virus types equally well (Cashdollar and Wymer, 2013), the recoveries reported in Table 1.3 represent a few selected studies which evaluated specific enteric virus types and filters. Upon review of the published literature for this investigation it was observed that the majority of investigators used electropositive filters supplied by either Nanoceram (Argonide, Sanford, Fl.) or Zeta Plus Virosorb 1MDS

(Cuno, Meriden, CT) (Karim et al. 2009). Once eluted, concentration of the eluent, to reduce its volume, whilst concentrating viral particles, may be employed through organic flocculation, filtration or precipitation methods. Briefly, organic flocculation involves the acidification of the eluent using HCl and slow stirring to permit the formation of small floc which is then centrifuged into a pellet and the supernatant discarded.

Precipitation involves adjusting eluates to a pH to 7.0, following with the addition of PEG and stirring at room temperature for up to two hours, after which precipitated virus is collected through centrifugation (Huang et al. 2000). In this study, flocculation was chosen as referenced in USEPA 1615.

Table 1.3. Filter options available to concentrate enteric viruses from waters

Filter type Elution Virus tested Recovery Citation

Glass wool- Glycine/beef extract Adenovirus, Coxsackievirus, 14-70% (Lambertini et al. electropositive pH 9.5 Echovirus, Norovirus and 2008) Poliovirus tested. Different recoveries depending on virus type

NanoCeram - Beef extract pH 9.5; Adenovirus, Coxsackievirus, 38% recovery (Karim et al. electropositive or salt-based Echovirus, MS2, Poliovirus, river water; 14- 2009); (Ikner et al. Rotavirus and Norovirus tested. 83% lab water 2011); (Pang et Different recoveries depending al. 2012) on virus type and source water type

Nitrocellulose - Sulfuric acid and Enterovirus 100% (Hsu et al. 2007) electronegative sodium hydroxide

Nitrocellulose Glycine and beef Enterovirus 7-21% (Hsu et al. 2007) electronegative extract pH 9.5

Electropositive Sulfuric acid and Enterovirus 0-28% (Hsu et al. 2007) membrane sodium hydroxide; or Glycine/beef extract at pH 9.5

Electronegative Sulfuric acid and Enterovirus 8-83% (Hsu et al. 2007) membrane sodium hydroxide, or Glycine/beef extract at pH 9.5

1.7 Detection and Enumeration of Norovirus and Enterovirus from source waters

Various approaches can be used to detect human enteric viruses in concentrated samples. Currently, the primary methodologies are based on either the detection of infectious viruses by propagation in cell culture or detection of viral genomes by molecular techniques such as quantitative polymerase chain reaction

(qPCR), reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for RNA viruses, or serological assays. Other methods such as, immunocapture PCR are presently being explored for the detection of viruses in water, but many researchers consider the molecular detection method in combination with culture-based isolation techniques as the current gold standard for virus detection (Baudart et al. 2000).

1.7.1 Cell culture

The gold standard for ascertaining viral infectivity is mammalian cell culture. This method uses specific cell lines which the virus can infect and, potentially, produce cytopathic effects which are observed as clearings or plaques in the host cell monolayer. Counting of the resulting plaques produces the plaque forming units

(PFUs). The observation of cytopathic effects produced in speciﬁc cell lines is not always possible as many epidemiologically important enteric viruses, such as human Norovirus, cannot be propagated in mammalian cell lines and for some enteric virus groups no single cell line will support the growth of all member viruses, such as within the human Enterovirus group (Bosch et al.2008). Research has recently been published showing human Norovirus can be cultured in the lab, however, this research is in early stages of development and no standardized methodology is currently available for detection in environmental samples

(Subba-Reddy et al. 2011). The principle drawbacks, however, even when cell lines are available are that this is not a simple or cost-effective technique as this method requires highly trained staff resources and materials to support the initial establishment and adaptation of cell lines and the virus of interest to culture conditions, and ongoing maintenance of the cell lines. Further, the length of time to verify a negative test may take several weeks to confirm (Fong and Lipp, 2005), and the method has been reported to underestimate the number of infectious viral particles in the environment as a result of low numbers within samples, difficulty in growing in culture, or replicate without producing CPE (Fong and Lipp, 2005).

1.7.2 Polymerase Chain Reaction (PCR)

The various detection methodologies that rely upon PCR require an initial isolation of viral nucleic acids from the samples of interest. A variety of commercial kits are available for nucleic acid extraction and purification. The majority of commercial kits are based on the method of Boom et al. (1990) whereby the cell/virus is lysed (guanidinium thiocyanate) and nucleic acids are captured on a column or silica beads. All nucleic acids from all cell types (such as plant, bacteria and viruses) that have been concentrated will be extracted simultaneously and may interfere with downstream assays. Approaches used in the extraction of nucleic acid from enteric viruses were evaluated by Griffin et al. (2014). They reported Norovirus mean recovery efficiencies of 87-90% for QIAmp DNA Blood Mini Kit, 98-100% and 75-94% respectively for

NucliSENS Magnetic Extraction using two different enzymes and primer probe preparations. Further, Ikner et al. (2013) reported the MOBIO PowerViral Environmental RNA/DNA Isolation kit to have higher viral nucleic acid concentrations and recoveries from environmental surface waters and sludges. Following nucleic acid extraction, quantitative PCR (qPCR) remains the method of choice for the detection of enteric viruses from environmental samples (Balasubramanian et al. 2016). qPCR is a molecular technique that quantiﬁes the numbers of copies of a target DNA sequence, genomic copies (GC), present in a sample.

Briefly, PCR uses the enzyme DNA polymerase to copy DNA or cDNA. When the target DNA or cDNA is present within the sample new DNA is created by the polymerase. Depending upon the PCR chemistry, the amplified DNA is detected using fluorescent dyes that can be detected and measured. qPCR assays have been developed for a number of enteric viruses causing gastroenteritis, some of which include: human

Norovirus (GI, GII); human Enterovirus; human Adenovirus; human Rotavirus; human Echovirus; human

Astrovirus; human Sapovirus; and human Bocavirus (Jiang et al. 2014; Liu et al. 2012).

Advantages of this technique include fast turn-around time (a couple of hours), it is easy to perform, provides real time data on synthesis of new amplicon molecules during the reaction (qPCR) and are performed within a closed tube thus minimizing risk of carry-over contamination. The qPCR assay utilizes small volumes (10 µL to 25 µL depending on protocol) for the amplification process therefore concentration methods that result in small volumes for the assay must be used. Additionally, the concentration and nucleic

acid methods should ensure the quality of the nucleic acid is not compromised by other compounds that will be simultaneously concentrated that may directly affect the analytical sensitivity of the assay or inhibit the amplification reaction of the qPCR assay. A number of steps and processes within the qPCR protocol can affect assay performance and impact results. The MIQE guidelines published by Bustin et al. (2009) provides a clear framework and standardized step wise approach to ensure results are high quality and reproducible. Environmental water samples are reflective of a snap shot in time and the composition of the matrix is highly dynamic, this is compounded by complex processing from sample concentration through to extraction and conversion of the enteric virus RNA into cDNA for the PCR assay. As such, qPCR experiments should incorporate the MIQE Guidelines into the experimental design and execution. qPCR provides sensitive detection and has been reported to detect enteric viruses (Rotavirus and Norovirus) as low as 1 cDNA copy per reaction (Pang et al. 2012). Additionally, multiplex real-time quantitative PCR assays can detect more than one viral target in a single assay through the use of multiple primer/probe sets that allow for simultaneous detection of major enteric viruses. One example is the simultaneous detection of the human enteric viruses Norovirus GII, rotavirus and adenovirus (strains 40 and 41) as described in Lee et al. (2016).

One significant limitation of the PCR assay is that it does not provide information on viral infectivity as it simply detects genomic material, providing an evaluation of the total viral abundance, including intact, infectious viruses and free viral nucleic acids. Thus results provide limited information when used in risk assessment applications, which require viable and infectious concentration values of the pathogen as model inputs. Research addressing this particular limitation of qPCR based assays includes studies on pre- treatment chemistries ahead of the nucleic acid extraction steps. Some of the best studied include pre- treating samples with nucleic acid intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide (PMA) so that free nucleic acids or nucleic acids from viral particles with damaged capsids, presumed inactivated and non-infectious, are unable to be amplified by PCR (McLellan et al. 2016). The application of EMA has been demonstrated to reduce false positive qPCR signals, thus increasing accuracy

of viral loading in complex environmental water matrices, but had limitations in distinguishing infectious and non-infectious virus after disinfection treatments involving heat or UV irradiation (Leifels et al. 2015).

1.7.3 Immunoassay or enzyme linked immunosorbent assay (ELISA)

Immunological tests such as enzymatic immunoassay or enzyme linked immunosorbent assay (ELISA) are commercially available kits (Invitrogen) for detection of key enteric viruses such as, Enterovirus (VP-1 polyclonal antibody) and Norovirus (capsid G2 Monoclonal antibody). ELISA detection systems are predominantly used for clinical samples, however in recent years they have been demonstrated effective for the testing of concentrated environmental water samples where detection limits have been reported between 10 3 and 106 virions/mL. Hryniszyn et al. (2013) tested Hepatitis A virus from untreated wastewater and reported a detection limit of 4 × 102 PFU/mL.

1.7.4 Integrated Cell Culture (ICC) PCR

Enteric viruses are not all culturable and some, such as Rotavirus and human Adenovirus are time consuming to cultivate in cell culture systems (Lee et al. 2004). The application of PCR reduces the time and therefore cost to detect the genetic material, but inhibitory factors from environmental samples can interfere with the assay and further, positive detection of nucleic acid material includes inactivated viruses.

Combining the feature of rapid detection of PCR and verification of infectivity of cell culture may increase sensitivity and reduce time to detection for those enteric viruses capable of propagation in a cell line. These methods have been combined and termed integrated cell culture and PCR (ICC_PCR). The combination of a cell culture step and subsequent detection by a molecular technique such as RT-PCR reduces the incubation periods and also allows the detection of viruses that grow without causing cytopathic effects.

Briefly, the integrated cell culture-PCR technique involves inoculation of the concentrated sample onto cell- line monolayers, which are then incubated for a minimum of 24 h; followed by RT-PCR analysis of the cell culture lysate. The virus does not need to produce CPE for detection as a sample of the culture is lysed and the lysate then used in the PCR assay-whereby the amplification and replication cycle begin to aid with detection of the target sequence.

This protocol has been reported as much faster than traditional cell culture; and the presence of infectious

Enterovirus can be confirmed within 24 hours instead of > 3 days and less labour intensive compared to the classical cell culturing (Reynolds et al. 1996). Detection reliability of indigenous viruses in environmental water samples by ICC-PCR can be restricted by the primer sets that are used (Lee and Jeong 2004). This approach has been used in viral particle inactivation studies to determine optimal disinfection strategies and doses for specific viral pathogens and used to detect infectious enteric viruses in river water, tap water, beach water, and wastewater effluent samples (Lee et al. 2005).

In summary, quantification by nucleic acid amplification has been successfully applied to the detection of enteric viruses in source waters intended for drinking water. The method is improved with the concentration of viral particles within natural source waters especially for those source waters with low concentration of the virus of interest. There remain methodological challenges to quantifying enteric viruses in source waters from the concentration step through to determining the number of infectious viruses present in a timely manner and these limitations pose challenges when attempting to apply models, such as QMRA, to enteric viruses. It has been reported that cell culture techniques are advancing for Norovirus, however, many other viruses within the enteric virus group remain unculturable, and while cell culture remains the gold standard for determining infectivity, it is time consuming and resource intensive, and as such, future study employing this technique alongside molecular pre-treatments (PMA and EMA) should be reviewed for Enterovirus and

Norovirus.

1.8 Qualitative Microbial Risk Assessment (QMRA)

The main risk to human health associated with drinking water consumption is drinking water contaminated with pathogenic microorganisms such as bacteria, viruses and protozoa. To reduce the risk of contaminated water, developed and many developing nations impose stringent regulations for treatment of drinking water and for the monitoring or verification testing of treatment and delivery system efficacy. Many developed nations such as Canada, the United States of America, and the United Kingdom have adopted

preventative multi-barrier approaches to ensure the delivery of safe drinking water through the establishment of multiple systems working in concert to form a safety net. Such barriers include, protection of the source water intended to be used, selection of appropriate treatment systems, management of the treatment and distribution systems, and the employment of qualified and trained personnel. To inform treatment strategies and operational management decisions, water managers need to be able to identify risks associated at each step in the processing of water for public consumption. Quantitative Microbial Risk

Assessment (QMRA) is one tool that can be applied by water managers to assess risk or probability of infection from pathogens such as enteric viruses. Risk assessment is a scientific based process involving four key steps: hazard identification; hazard characterization; exposure assessment; and risk characterization (Hunter et al. 2003). In the regulatory context, QMRA is used to determine potential health risk to consumers of waterborne pathogens based on source water concentrations after treatment. Dunn et al. (2014) reported variability in microbial risk assessment frameworks and management tools across international agencies, noting that method implementation lacks standardization when QMRA approaches are adopted. Outbreaks from waterborne enteric viruses have been reported from drinking water systems where conventional water treatment processes meet treatment targets. Legislated or regulated requirements to perform verification checks on the quality of source waters entering and leaving treatment processes, for human enteric viruses do not currently exist. Pathogen specific testing of source waters by cell culture and molecular assay has been considered cost prohibitive; resource intense as testing requires specialized trained personnel to maintain cell cultures and to operate sophisticated molecular detection equipment; and may lack sensitivity; primarily as a result of the need to filter large volumes of water.

Improved and novel microbiological detection methods are critical in water characterization studies to predict the occurrence of viral pathogens. There is a paucity of published research on application of QMRA for enteric viral pathogens in source waters to assess effectiveness of drinking water treatment systems.

Chigor et al. (2014) employed a four-step static QMRA to six river sites that were sampled for Enterovirus,

Rotaviruses, Adenoviruses and Hepatitis A virus that indicated annual risks projected by the model are higher than South Africa’s recommended 1 in 10,000 probability of infection.

To determine pathogen risk, there is a need to characterize source waters for pathogens or surrogates representing bacterial, protozoan and viral groups. Without quantitative data, risk cannot be adequately portrayed or estimated (Payment, 2014). Characterization studies depend on sampling programs focused to include times when treatment challenge would be expected (precipitation and runoff events) to better characterize source waters and threats to existing water treatment systems and processes.

QMRA is beginning to appear in regulatory frameworks. Health Canada has developed guidelines and a mathematical model to quantitatively assess the potential microbiological risks – derive disease burden estimates from selected pathogens (viral, bacterial and protozoan) associated with drinking water treatment systems (Health Canada, 2012). The Dutch Drinking Water Act (2001) has enacted legal requirements for water utilities to perform QMRA for sources of water intended for drinking (surface water and vulnerable groundwater) every three years and requires F-specific or somatic bacteriophages as indicators of

Enterovirus (Schijven et al. 2011). QMRA investigations are emerging, especially noteworthy are recent publications in recycled wastewater and irrigation waters as they are becoming a crucial contribution to water supply strategies in times of water quantity and conservation and climate change (Schoen et al.

2017). Australia employs QMRA to regulate water quality from recycling schemes for non-potable reuse presently, but is reviewing regulations under the national drinking water guidelines to consider its application for potable water supplies and treatment systems (Bichai and Smeets, 2013).

Although risk assessment, with data on virus concentrations in the source water and the treatment efficiency, is an important tool to estimate the possible risk of infection, it is necessary to obtain more information about the occurrence and prevalence of enteric viruses (Norovirus and Enterovirus) present in source waters intended for drinking water. Currently, in Ontario, characterization studies of source waters for enteric viruses is not mandated by regulation, and very few jurisdictions perform surveillance and testing for these enteric virus types in water (Appendix 6 – jurisdictional scan on source water testing for viral pathogens).

1.9 Hypothesis

Human Norovirus GII and human Enterovirus occurrence in the environment, particularly in sources of drinking water, is needed to understand the risk to the population served by these sources. Enteric virus loading and spatial temporal occurrence is important information needed to make informed decisions on the approach and implementation of prevention, control and treatment strategies for effective water treatment facility operations.

It was hypothesized that human Norovirus GII and human Enterovirus within an urban watershed would be detectable in the concentrated raw river water samples (RW) and may correlate with higher turbidity, precipitation events and river flow measurements, as the river source is impacted by anthropogenic activities that may contribute viral pathogen loading to water, including effluent from large urban wastewater treatment facilities, and rural septic systems. Further, it was assumed that human Norovirus GII and human

Enterovirus detection would correspond with late fall to early spring, and mid-summer to early fall respectively, mirroring monitored and reported community associated illness and outbreaks.

It was also postulated that human Norovirus GII and human Enterovirus within the same urban watershed may be detectable in the concentrated raw ground water samples (GW) as a result of virus entering the shallow upstream aquifer located directly under the river, and that Norovirus GII and Enterovirus if detected in these ground water samples, may also demonstrate seasonal occurrence as hypothesized for river water samples. Similarly, it was theorized that the detection of Norovirus GII and Enterovirus in the GW samples may show a correlation with higher turbidity, precipitation events and river flow measurements as a result of the shallow aquifer’s hydraulic connection to the river source.

Objective

1. Using the USEPA1615 method, sample source waters (surface water and ground water sites) for 16

consecutive months on a routine sampling schedule, and includes precipitation events, to quantify

human Norovirus and human Enterovirus in environmental sample concentrates. Wastewater

treatment effluent will be sampled in the winter and the fall to ascertain community presence of human Norovirus and human Enterovirus.

Chapter 2: Materials and Methods

2.1 Norovirus and Enterovirus Nucleic Material

Human Norovirus and human Enterovirus RNA purified from clinical samples were generously provided by

Dr. P. Jayaratne (Hamilton Regional Laboratory Medicine Program, Hamilton, ON, Canada).

2.2 Source Water Sampling

2.2.1 Water Sample Site Locations and Collection Technique

Raw water samples from two locations in southwestern Ontario Canada’s Grand River watershed were collected (Section 2.3.2) over a 16 month period from December 2014 to April 2016. The study sites were selected to allow testing of groundwater and surface water sites. The municipality requested that the sites be anonymized; as such sites were coded GW (groundwater) and RW (river water), respectfully.

2.2.2 Ground Water (GW) Site Description

The Grand River Watershed has 81 wells within 23 well fields and a surface water intake that serve the Tri- city area of Kitchener, Waterloo and Cambridge Ontario. The source of GW selected for this study has been deemed ground water under the direct influence of surface water (GUDI) with effective filtration by the

Ontario Ministry of the Environment and Climate Change. This GW well was constructed in 1987 and is outfitted with a stainless steel liner to 29m below ground surface(approximately), and can supply up to 25 L

/s without impact to water quality. Although the well has a rated flow of 75 L/s according to municipal water supply engineering reports (Stantec, 2014). These reports also document the untreated (raw) GW source located within the urban boundary of Cambridge, Ontario to contain concentrations less than the Ontario

Drinking Water aesthetic objectives and guidelines for sodium (200 mg/L), iron (0.3 mg/L), manganese (0.05 mg/L) and sulphate (500 mg/L) (Stantec, 2014).

2.2.3 River Water Site Description

Surface water was obtained from an upstream Grand River source within the urban boundary of Waterloo,

Ontario. The physical geology, hydrology and water characteristics of the Grand River have been well documented by the Grand River Conservation Authority (GRCA, 2011) as part of Ontario’s legislated requirement (Clean Water Act, 2006) for the development of source water protection plans and the establishment of source water protection zones. Briefly, water quality over the 300 km length of the Grand

River is influenced by ground water inputs, agriculture, wastewater inputs from the tri-cities of Waterloo,

Kitchener and Cambridge, surface runoff and storm water. The Grand River watershed covers a large area

(6,800 square kilometers (GRCA 2017, https://www.grandriver.ca/en/our-watershed/Our-Watershed.aspx) running south from its source near Wareham Ontario into the north shore of Lake Erie, and is comprised of overlapping areas of glacial till and moraine. Water quantity and quality are largely controlled through manmade water control structures (dams and reservoirs). Water quality of the Grand River is routinely monitored through multiple agencies and organizations and is perhaps one of the most studied watersheds in Ontario. The Grand River sees elevated levels of sodium (groundwater influence and winter deicing infiltration) and shows variable concentrations of nutrients (phosphorous, nitrates), turbidity and microbiological activity that change with the seasons (GRCA, 2008)

Grand River flows are controlled through engineered structures, as such are variable across the length of the river. Base dry weather flows fluctuate depending on precipitation; 2016 was reported as unusually dry

(GRCA, 2016).

2.2.4 Wastewater (WW)

There are 29 municipal secondary and tertiary wastewater treatment facilities discharging to the Grand

River year-round (GRCA 2009). Wastewater samples (raw untreated influent and pre/post ultraviolet treatment) were collected from one municipal wastewater treatment facility (WWTF) within the urban boundaries of Kitchener-Waterloo, Ontario and tested for the presence of human Norovirus and human

Enterovirus to provide information on community viral particle numbers from the wastewater collection and

treatment system that could bypass treatment and impact water quality within the Grand River and downstream groundwater sources that may be influenced by the river water of the Grand River.

2.3 Sampling Plan

A total of 21 grab samples were collected at the GW site and 22 grab samples from the river water site.

Two raw wastewater influent samples (WW) were collected in the winter of 2014/2015 and 2015/2016, respectively and two pre- and post-UV treatment were collected, one in the winter of 2014 and a second in the fall of 2015.

Samples were collected between December 2014 and April 2016. Over the study period, samples were not taken in August and December of 2015. Sampling frequency occurred monthly from January 2015 through to July 2015 and was increased to bi-weekly September 2015 to April 2016. Additionally, samples were collected during precipitation and snow melt events. Precipitation events were defined as those greater than or equal to 15 mm of rain. No specific flow rate was defined for snow melt events; instead an observational approach was applied based on observed elevated temperatures, visual confirmation of reduced snow cover and increased depth and flow rates. Appendix 1 contains the detailed sampling record.

Seasonality was investigated by analysing the occurrence of viral particles within the four seasons.

Seasonality start dates were assigned based on the 2015 and 2016 equinox and solstice dates

(http://aa.usno.navy.mil/data/docs/EarthSeasons.php last accessed September 2016).

Seasonal prevalence and variability of human Norovirus GII and human Enterovirus was determined based on monthly and biweekly sample collections between December 2014 and April 2016 as shown in Table 2.1.

Table 2.1 Sample collection between December 2014 and April 2016 Season Start date Number of Collection Details sample collections Winter 2015 December 21, 9 One wastewater raw influent in December 2014 2014; One GW and one RW in January 2015; One GW and one RW in February 2015; and Two GW and two RW in March 2015 Spring 2015 March 20, 2015 6 One GW and RW in April 2015 One GW and RW in May 2015 One GW and one RW in June 2015 Summer 2015 June 21, 2015 2 One GW and RW in July 2015 One GW and RW in September 2015 Fall 2015 September 23, 11 One GW and RW in September 2015; 2015 Two GW and three RW in October 2015 Two GW and two RW in November 2015 Winter 2016 December 21, 13 No GW or RW in December 2015 2015 Six GW and RW in January 2016 Five GW and RW in February 2016 Spring 2016 March 20, 2016 3 One GW and RW in March 2016 Two GW and RW in April 2016

2.3.1 River conditions and water quality observations

Samples were collected on a scheduled basis to facilitate access to sampling locations. Attempts were made to sample consistently and additionally during precipitation or snow melt events. Rainfall data for the

Region were extracted from Environment Canada’s web site (www.climate.weatheroffice.gc.ca). River flow and ambient air temperature data were extracted from the Grand River Conservation Authority’s website

(https://www.grandriver.ca/en/our-watershed/River-data.aspx?_mid_=1032).

At the sites, additional water quality parameter results were obtained from the municipality, including turbidity, conductivity, temperature, total suspended solids, and bacterial indicators; total coliform, and E. coli.

Physical and chemical data were acquired from onsite inline data logging units connected to the municipality’s water treatment operations computer system. Bacterial indicator data was acquired from the

municipalities ISO17025 accredited and Ontario licensed drinking water testing laboratory. Bacterial results obtained from the groundwater site from December 1, 2014 to April 17, 2016 were based on the Presence or Absence test (APHA Standard Method 9221.D) method, as per Ontario Regulation 170/03 (O.Reg.

170/03), for total coliform and E. coli on raw ground water sources. River water samples from December 1 to February 5, 2016 were analyzed using the membrane filtration method using M-Endo (APHA Standard

Method 9222B) and MFc-BCIG (APHA Standard Method 9222D) agars, as per O.Reg. 170/03 for total coliform and E. coli on raw surface water sources. Data can be found in Appendix 2.

2.3.2 Sampling Technique

Water samples were collected as outlined in the United States Environment Protection Agency (USEPA)

Method 1615, “Measurement of human Enterovirus and human Norovirus Occurrence in Water by Culture and RT-qPCR”, using apparatus as seen in Figure 2.2 (USEPA 2012). The filter apparatus was set up at each sample site as specified in USEPA1615 (Figure 2.2). A flow meter was connected to the discharge line of the filter housing to monitor and record the total volume of water that passed through the filter and housing assembly.

Figure 2.1 The virus sampling apparatus at the raw river water intake sampling line

Briefly, prior to water sample collection, the filter housing and tubing were cleaned, then disinfected

by treatment with 0.525% sodium hypochlorite for 30 min, followed by a rinse with sterilized reverse osmosis

Flow meter laboratory grade water and sterile dechlorination solution containing sodium thiosulfate (50 mL of 1 M

sodium thiosulfate), with open ends wrapped in sterile foil or petri film. At each sampling site a unique filter

housing and new sterile filter were used. At the sampling site and prior to adding the Nanoceram filter

(Argonide, Sanford, Fl), the filter housing was connected to a tap or pipe and flushed with 75L of raw source

water. After flushing, a sterile Nanoceram filter was placed into the housing for sample collection. For GW

sampling, either a minimum of 1250 L or maximum of 1500 L (quantity sampled was dependent on time

available at the site) of raw source water was collected using a flow rate of 10 L/min through the filter. For

the river water sample site, the raw source water volumes filtered varied as a result of water turbidity at the

time of sampling. Flow rates at this sampling site varied from 0.5 L/min up to 4 L/min, as such sampling was

capped at two hours resulting in sample volumes ranging from 38 L up to 450 L with a median volume of

195 L filtered at this site over the study period. After sample collection, the filters were kept in their

respective housings and transported on ice to the laboratory, then stored at 40C until processed.

Processing of the filters began within 72 h of sampling.

2.4 Concentration of water samples

Filters were eluted and concentrated according to USEPA Method 1615 with minor modifications (Figure

2.3). Briefly, each sample filter was eluted with 1 L of sterile 1.5% beef extract containing 0.05 M glycine at

pH 9.0. Using a peristaltic pump to create positive pressure, a 500 mL aliquot of the beef extract was

pumped into the filter housing and held in contact with the filter for 1 minute before eluting from the filter at a

rate of 10 L/min into a sterile 2 L flask. The second application of 500 mL beef extract was then pumped

into the housing, and allowed a contact time of 15 min prior to the second elution into the collection vessel.

Figure 2.2 A schematic diagram showing the stepwise approach used in this study from field sample collection to qPCR assay

Figure 2.3 The laboratory sample elution apparatus and set-up

After elution the pH of the eluate was dropped to 3.5 by slowly adding 1.2 N HCl. The eluent was mixed slowly for 30 min to facilitate flocculation. The samples were then centrifuged for 30 min at 2,500 x g at 40C. After centrifugation the supernatant was discarded. The resulting pellet was re-suspended in 30 mL 0.15 M sodium phosphate (pH 9.0) in the centrifuge bottle by stirring with a stir bar at low speed for 10 min. After removing the stir bar, the sample was centrifuged at 10,000 x g at 40C for 15 min. The supernatant was collected into a sterile tube and its pH adjusted to 7 to 7.5 with 1.2 M HCl. The supernatant (referred to as the secondary concentrate) was subsequently filter-sterilized first through a 0.45 µm sterile syringe filter and then a 0.22 µm sterile syringe filter (VWR Pall Syringe Filter 28145-501). The sample was split into two 15 mL volumes. One 15 mL aliquot was further concentrated (tertiary concentrate) using a centrifugal ultrafilter (Vivaspin® Turbo 15, Sartorius-Stedim Biotech GmbH, Gottingen,

Germany) to a final volume of 400 µL. A 200 µL subsample of the tertiary concentrate was used for nucleic acid extraction and the remaining 200 µL subsample was either processed as a duplicate sample or stored at -800C for future culturing work. The remaining 15 mL volume of secondary concentrate was stored at -800C for future study.

2.5 Nucleic acid extraction

All secondary and tertiary concentrates were extracted within 24 h of elution. Samples from

January 15 to March 21, 2015 were extracted using the viral DNA and RNA extraction and purification kit from QIAamp® MinElute virus Spin Kit (57704 Qiagen, Hilden, Germany).

Samples from April 15, 2015 to April 17, 2016 were extracted using MOBIO PowerViral

Environmental RNA/DNA Isolation kit (MoBio Laboratories Inc., West Carlsbad, California,

USA). The manufacturer’s instructions were followed for each kit without deviation. Briefly, 200

µL of secondary concentrate or 200 µL of tertiary concentrate sample were used. A final

RNA/DNA extracted sample of 100 µL in RNase/DNase free water was collected and stored at - 41

800C until reverse transcription could be performed. To conserve sample volumes and maintain sample integrity, DNA/RNA concentrations were not determined prior to storage.

2.6 Reverse Transcription and Quantitative Polymerase Chain Reaction

(RT-qPCR)

The (RT)-qPCR procedures for detection of Norovirus and Enterovirus followed the protocol listed in the USEPA 1615 method, with some minor modifications. In this study we utilized the iScript™ cDNA synthesis kit (Biorad) as per the manufacturer’s protocol. Briefly, a total assay volume of 20 µL contained 6.7 µL of RNA extract, 1 µL reverse transcriptase, 4 µL iScript ™ reaction mix and 8.3 µL nuclease-free water. Reverse transcription was carried out as per iScript™ protocol, using a BIORAD S1000™ thermal cycler, 250C for 5 min, 420C for 30 min, followed by 850C for 5 min and then held at 40C. A no-template control and no-reverse transcriptase control were also prepared with each batch of cDNA synthesis reactions.

Samples were kept at -200C until qPCR could be performed.

2.7 Preparation of Plasmid Standards (Standard curve)

Norovirus, Enterovirus, and MS2 cDNA were amplified by PCR following the GOTAQ® Hot Start

Green Master Mix protocol (Promega Corporation, Madison, WI). Primers and probe (see Table

2.2) were used to target the conserved capsid gene ORF2 for human Norovirus; the 5' NCR gene for human Enterovirus; and the 77 bp segment of the gene encoding the coat protein (or A protein) for MS2 (Monpoeho et al. 2001; O’Connell and Bucher, 2006). Amplified DNA was cloned into separate plasmid vectors using the pGEM®-TEasy Vector System (Promega

Corporation, Madison, WI, USA). Vectors were then transformed separately into competent

Escherichia coli JM109 cells and selected based on ampicillin resistance and colour development on LB agar plates containing 50 µg/mL ampicillin, IPTG and X-gal. Putative E. coli transformants were screened for the presence of target markers by colony PCR and gel electrophoresis. Plasmids were extracted using the Promega Wizard® Plus SV Minipreps DNA

Purification System as directed. The concentration and absorbance readings of the purified plasmids were measured using a Nanodrop1000 spectrophotometer (Thermofisher, Wilmington,

DE). Copy numbers were determined following the instructions given in the following website: http://scienceprimer.com/copy-number-calculator-for-realtime-pcr.

Plasmid stocks for each of the three plasmid types were aliquoted into single use volumes and stored individually at -800C. Plasmid stocks for each of the standards (MS2, human Norovirus and Enterovirus) were serially diluted (10-fold dilutions) to create working dilutions from 2.5 x

109 to 2.5 GC/µL; Plasmid working dilutions for each of the three plasmid types were stored at -

200C and made fresh every two weeks from the plasmid stock.

2.8 Method Performance Spike Evaluation with MS2 Bacteriophage

To test viral particle recovery of the method (from elution through to PCR assay), MS2 phage

(1010 PFU/mL) was spiked into 10 L of both raw river water and raw ground water to a final concentration of 107 PFU/mL. Following field sampling at both groundwater and surface water sites, an additional 10L of unfiltered water were collected from the inlet at each site in separate sterile carboys that were then transported to the lab for spiking prior to filtration through the

Nanoceram filter. Spiked samples were processed in the laboratory as previously described

(Sections 2.4 to 2.6). Spiked samples (raw unfiltered source, and raw filtered) were processed

(direct RNA extraction and cDNA production) as described above and enumerated by RT-qPCR to determine percent recovery of spiked MS2. The qPCR assays were carried out using the

Applied Biosystem QuantStudio™ real-time PCR system as follows: 6 µL of MS2 spiked sample cDNA was added to a microplate well containing 10.0 µL of SSoFast ™Evagreen

Supermix® (BioRad), and 2 µL each of forward and reverse primers (400 nM each, Table 2.3) targeting the gene encoding MS2 assembly protein (O’Connell and Bucher, 2006) for a total volume of 20 µL per reaction. Reactions were carried out in duplicate, the cycling protocol was as follows: 95°C for three min, then 95°C for 10s followed by annealing at 60°C for 15s

(repeated 40 times), and a melt curve from 65°C to 95°C, with readings taken at 0.5°C intervals to determine if non-target material was present. Samples and plasmid standards were run in duplicate; and positive controls and no template controls were included for each assay.

For data analysis, the baseline threshold was determined by the Applied Biosystem software.

Only standard curves with efficiencies between 90 to 105% and R2 values between 0.950 and

0.999 were used, as indicators of consistent product amplification between assays.

2.9 Determination of qPCR Inhibition using Standard Plasmids

Inhibition dilution tests were performed on every field sample to evaluate matrix interference with the qPCR assays. Methods for the preparation and application of plasmid standards for evaluating inhibition have been detailed previously (Lee et al. 2016). In short, a series of sample dilutions for each cDNA sample were prepared, diluted ½; ¼; and ⅛ with TE buffer.

Following the USEPA1615 protocol, standard plasmids with known concentration, 2.5x10 6 copies, for Enterovirus and Norovirus genes were used as template DNA in the tests. Each plasmid standard was used in separate sets of diluted cDNAs. The Ct values for the

Enterovirus and Norovirus plasmid concentration in Tris-EDTA buffer alone were 24 and 33, respectively in this investigation. The inhibition qPCR assays were carried out using the Applied

Biosystem QuantStudio™ real-time PCR system. One µL of working stock plasmid (2.5 x 106 of

either Enterovirus or Norovirus GII) and 5 µL of diluted sample cDNA were added to a microplate well containing 15 µL of MasterMix (Table 2.2), and 4 µL molecular grade water, for a total volume of 25.0 µL per reaction. PCR reactions were carried out in duplicate as per

USEPA1615 protocol: 1 cycle @ 950C for 10 min, 45 cycles @950C for 15 s and 600C for 60 s.

Positive controls and no template controls were also prepared with each PCR run. The Ct values of the spiked samples were then compared to that of the baseline control.

Table 2.2 PCR Master Mix for Norovirus and Enterovirus Assay and PCR conditions

Reagent Quantity

2X PCR Mix (Applied Biosystems) 12.5 µL per reaction 2XTAQMAN

Water (molecular grade) 0.48 µL per reaction

Primer F* 1 µL per reaction (NorGIIF 500nM) (EntF 300 nM)

Primer R* 1 µL per reaction (NorGIIR 900 nM) (EntR 900 nM)

Probe* 0.025 µL per reaction (NorGIIP 250 nM) (EntP 100 nM)

Thermocycler conditions 1 cycle @ 950C for 10 min, 45 cycles @950C for 15 s and 600C for 60 s.(USEPA1615 protocol)

TOTAL VOLUME 15.00 µL

*see Table 2.3 for primer and probe sequences/targets

Samples that exhibited significant deviations from the Ct value of the positive control (> 1.0Ct) were considered inhibited. Samples that showed inhibition at dilution were further diluted prior

to qPCR to, and for subsequent quantification assays. Table of inhibition study results for

all processed samples from river water and ground water sites can be found in Appendix 3.

2.10 Quantitative PCR Assay for Norovirus and Enterovirus qPCR was used to provide a quantitative estimate of Enterovirus and Norovirus GII genomic copies per liter (GC/L). qPCR assay calibration was conducted using quantification of plasmid standards containing the target gene sequences. Plasmids were prepared in a series of 10-fold dilutions as stated above (Section 2.7). The serially diluted plasmid standards, 2.5x108 to

2.5x103 GC/µL, were prepared in duplicate for each qPCR run. In brief, 10 µL of sample cDNA were added to a microplate well containing 15.0 µL of Master Mix (Norovirus, or Enterovirus mix) (Table 2.1) for a total volume of 25.0 µL per reaction. Samples were run in duplicate and every qPCR plate was run with positive and negative controls. Plasmid standard dilutions served as positive controls for each PCR run. PCR assays were deemed successful when positive controls reproduced the known threshold cycles (Ct) along the dilution series. Negative controls (no cDNA template) were set up for each PCR run by adding TE buffer or molecular grade water to the PCR Master Mix. Norovirus and Enterovirus primer and probe sets were evaluated for cross reactivity using Adenovirus DNA (strain Dugan, ATCC VR-931), and

Rotavirus cDNA strain Wa. No amplification was observed for these samples with the PCR assays.

Table 2.3 Primers and TaqMan® Probes for Virus Detection by RT-qPCR

Virus Primer Oligo Sequences Sequence Tm PCR Reference (F and R) (5’ to 3’) target (°C) product or Probe (bp) (P) Norovirus NorGIIF CCAATGTTCAG 59.1 92 Lee et al. GII.4 ATGGATGAGAT 2016 TCTC NorGIIR TCGACGCCATC 57-60 Kageyama et TTCATTCACA) al. 2003

NorGIIP 6FAM- ORF2 USEPA1615, AGCACGTGGGA Butot, 2010 GGGCGATCG- TAMRA

Enterovirus EntF CCTCCGGCCCC 58.0 196 DeLeon et al. TGAATG 1990 EntR ACCGGATGGCC 56.0 DeLeon et al. AATCCAA 1990 EntP 6FAM- 5′NCR Monpoeho et CGGAACCGACT al. 2000 ACTTTGGGTGT CCGT-TAMRA MS2 MSF GTCGCGGTAAT Assembly 58-60 150 O’Connell et TGGCGC protein (A- al. 2006 protein) MSR GGCCACGTGTT O’Connell et TTGATCGA al. 2006 Universal Probe (Biorad) FAM Fluorescein reporter dye. TAMRA™ quencher

2.11 Statistical evaluation

Each sample was analyzed in duplicate to calculate a mean. All statistical analysis was performed on the back-calculated values from the qPCR results using MS Excel version 2010.

Data was divided into two sets and analyzed. Data set 1 included GC/L values greater than or equal to 3 GC per PCR reaction , (the lower limit of RT-qPCR quantification as defined by

Bustin et al. 2009), whereas data set 2 included all positive PCR detections (presence). To

compare mean virus counts with physio-chemical and biological variables, a Pearson correlation and a logistic regression were performed with significance set at P≤ 0.05.

Chapter 3: Results

3.1 MS2 recovery

MS2 was selected as a representative enteric virus surrogate for the purpose of determining process recoveries from environmental waters, since MS2 is known to be morphologically similar to mammalian non-enveloped viruses such as Norovirus and Enterovirus (Bae et al.

2008). MS2 recoveries for the VIRADEL procedure were determined to be 5 to 30% for undiluted secondary concentrate samples and as low as 1 to 3% for undiluted tertiary concentrate samples. This test was used to determine the efficiency of in-lab processing and values pertain to the in-lab recovery of MS2 spiked into 10L of environmental water (either river or groundwater, final concentration 107 PFU/mL) to evaluate filtration through elution and re- concentration, cited above, for the processing of field samples. The USEPA Method 1615 states the acceptance criterion for QC and matrix spikes for the molecular procedure is a recovery of 5–200%.

River water matrix: The secondary recovery at 30% was within the acceptable range required by the USEPA method. The tertiary recovery of 3%, however, did not meet the requirements of the USEPA method. To better understand inhibitory effects on the spike within environmental waters, future studies could consider performing the recovery test with a series of diluted spiked samples.

Ground water matrix: The secondary concentrate recovered only 5% of MS2 from the MS2 spiked groundwater sample. While the tertiary recovery of MS2 from the secondary sample processed was 38%. For the groundwater samples the tertiary concentration provided recoveries within the acceptable range for the USEPA method.

Low recovery could be the result of lost nucleic acid at a number of key junctures within the processing protocol. Recovery for this process is on the lower end of the acceptable range and could be attributed to nucleic acid loss during secondary and tertiary concentration; specifically viral particles may pass through the tertiary filter or remain stuck to the filter. Additional losses may have been realized during transfer into the RNA extraction substrate or during RNA extraction, as not all secondary and tertiary concentrate volume was utilized in the RNA extraction step, nor was all RNA applied in the RNA extraction process used. A second extraction could be performed on the remaining 150 μL to ensure the 200 μL volume is completely processed and combined. Similarly, during reverse transcription, a small proportion of the extracted RNA volume was transcribed (6 µL) and subsequently used to perform the qPCR. Although samples were well mixed prior to micro pipetting – target viral particles could be missed if the quantity of virus was low.

Additionally, viral particles may not be evenly distributed within a sample and viral particles may have been missed during transfer pipetting of such small volumes throughout laboratory processing.

Spiked samples in this study were not diluted prior to qPCR assay, as a high concentration of

MS2 was employed to overcome any inhibitory effects which may have been present within the untreated environmental waters. However, inhibitory substances within source waters such as humic acids and reagents used to process source waters are known to exhibit some inhibitory effects on PCR assays (Abbaszadegan et al. 1992; Lee et al, 2016; Prevost et al. (2016);

Schwab et al.1993). Future testing could include a check for PCR inhibition by performing inhibition studies on spiked samples, which then could be reduced by diluting the spikes prior to molecular assay, thus potentially improving reported recoveries. In this study recovery testing was performed at the onset of the investigation in the winter. Additionally, based on the results,

recovery testing should be performed on source water for each season to understand if seasonal impacts on environmental waters affect test method recoveries.

3.2 PCR Inhibition by Environmental Samples

Nucleic acid extracts of environmental samples are known to exhibit inhibitory effects on the

PCR assay. Thus, for this study PCR inhibition was examined on nucleic extracts from all water samples. Norovirus GII and Enterovirus standard plasmids were spiked into each environmental sample dilution at a concentration expected to be higher than values in the environmental samples (2.5x106 genome copy per µL). This provided inhibition data specifically for the target sequences of interest. 22 river water samples and 21 groundwater samples were evaluated at the qPCR stage for inhibition. For each sample type, nucleic acid extractions from both secondary and tertiary concentration steps were assayed by PCR for the presence of inhibition using both human Norovirus GII and human Enterovirus primer/probe sets. Each sample was diluted, two-fold serial dilutions, up to 1:8. Samples were deemed inhibited if they did not produce a Ct value within 1 Ct of the target baseline control (mean value of duplicates)

(Cao et al. 2012). Samples that exhibited inhibition at 1:8 were further diluted to 1:16 for qPCR.

Susceptibility to inhibition differed between the Norovirus and Enterovirus primer probe sets.

Inhibition was observed in 100% of undiluted raw river water samples, for both Norovirus and

Enterovirus primer probe sets. 100% of undiluted raw groundwater samples showed inhibition when tested with the Enterovirus primer/probe set (Appendix 3). Eighteen of the twenty one groundwater samples exhibited inhibition with the Norovirus primer/probe set, and only one of those was positive for the detection of Norovirus (quantity below the limit of quantification

(LOQ)). Primer/probe sets and sample specific matrix inhibition have been reported previously;

they support the importance of checking each sample with each primer probe for qPCR inhibition to improve accuracy of reported results (Borchardt et al. 2003).

PCR inhibition was mostly removed at dilutions of 1:8 for GW samples and 1:16 for river water samples. The USEPA recommends dilutions up to 1:25 for surface waters. In this study dilutions were taken to 1:16 for those samples displaying inhibition still at 1:8 to minimize dilution of nucleic acid material. Ten river water samples were selected that detected only one of the virus types (either Norovirus or Enterovirus, but not both) at a 1:16 dilution, and were further diluted to 1:32 to see if further dilution would detect the other virus type. Of the ten river water samples, two samples showed a positive qPCR reaction at a Ct of 38.1 and 37.98 (≥

LOQ), respectively, for Norovirus, and two samples showed a positive qPCR reaction at a Ct of

43.48 and 44.45 (< LOQ) respectively for Enterovirus when further diluted to 1:32.

Molecular methods have been reported to be sensitive to environmental inhibitors (Borchardt et al. 2003). It was observed that all field samples from this study exhibited inhibition of the qPCR assay, and thus required dilution of the nucleic acid extracts. In conducting the inhibition tests it was noted that amplification efficiency differed between the Enterovirus and Norovirus sets of primers and probes used in this investigation. In this study it was observed that samples for the detection of Norovirus GII, overall, required less dilution prior to PCR assay than that of the same source waters for detection of Enterovirus using the Pan-Enterovirus primer and probe set that could detect multiple serotypes within the Enterovirus genus such as Enterovirus A, B, C and D (Appendix 3), which suggests the Enterovirus primer/probe set is more sensitive to compounds within the water sample or assay reagents. Inhibition data (Appendix 3) was also examined to determine if the inhibition test on samples going forward could be performed solely using the more sensitive primer probe set, the Enterovirus primer probe combination, to optimize resources and reduce the consumption of reagents. It was found that this approach would not be advantageous for future sample testing, as field samples would be diluted further

than necessary therefore impacting the sensitivity of detection for human Norovirus GII and may result in the reporting of non-detects. It was observed from the dataset that inhibition testing for each primer probe set for every sample should be practiced in future studies.

3.3 Quantification of Norovirus and Enterovirus by qPCR

Quantification of Norovirus and Enterovirus genomes present in the field samples was based on a plasmid standard curve that ranged from 3 to 3 x 106 genomic copies. The limit of quantification (LOQ) was set at 3 GC per PCR reaction based on the specific standards described in Bustin et al. (2009). As such, experimental samples with values at or above 3 were considered positive for Norovirus or Enterovirus, and were subsequently quantified.

Each set of assays was run with a six point plasmid standard curve containing the target sequence. Standard curves for all qPCR assays were deemed acceptable when the correlation coefficient (R2) of the standard curve was ≥0.97 and the standard deviation was <0.25 (USEPA

1615). Primers and probes were carefully selected to detect only the nucleic acids of the specific viruses being analyzed, and none of the primer/probe combinations cross-reacted when tested against Adenovirus and Rotavirus. Sample reactions were run in duplicate (technical replicates) with positive and no-template controls on each plate. PCR assays employ a small proportion of the original sample and therefore represent a very small percentage of the original field sample. Ten µL of cDNA used in this study has been diluted; dilution factors are variable, dependant on volumes generated during secondary and tertiary concentration processing steps.

Ten µL of cDNA, the maximum volume of template that could be added to the assay, was added to each reaction to increase probability of detecting the target previously diluted to reduce inhibitory effects. The mean value of qPCR genomic copies was calculated to genome copies per liter (GC/L) of raw sampled/filtered water (Appendix 5). In this investigation qPCR assays

demonstrated reaction efficiencies (yield) between 78% and 118%. qPCR reactions demonstrating 90% to 110% reaction efficiency are deemed acceptable within the USEPA1615 method. Efficiencies above 110% may be indicative of possible inhibition in the real time reaction. In this study samples were diluted to try to reduce inhibitory effects and improve efficiency. PCR assays below 90% efficiency were run again to optimize reactions, runs whose efficiencies were above 110% were accepted, as samples were already diluted to accommodate for inhibition and further dilution to reduce the upper limit of efficiency from 118% to 110% may have resulted in a further reduction in sensitivity of detection.

Secondary and tertiary concentrates for each sample type were assayed (Figure 2.3). Twenty- two river water secondary concentrate and 22 river water tertiary concentrate samples, and 21 groundwater secondary concentrate and 21 groundwater tertiary concentrate samples in total were assayed by qPCR for the presence of both Norovirus and Enterovirus. Of the river water concentrates assayed, 12 samples were positive for Norovirus (27%) whereas only 5 (11%) were positive for Enterovirus. Of the groundwater concentrates analyzed, 11 were positive for

Norovirus (26%) whereas 12 (29%) were positive for Enterovirus. An observation from this study was the influence of source water type on detection from secondary and tertiary concentrates. We observed an increase in detection of viral particles for the river water secondary concentrate showing that tertiary concentration for this sample type may lead to lower detection rates based on inhibition or viral particle loss during lab processing. For raw groundwater samples, the opposite was observed, in that a slight increase in detection of viral particles was seen when samples were further processed into a more concentrated tertiary concentrate.

Virus counts (GC/L) for human Norovirus and human Enterovirus from both GW and river water sample sites for all samples analyzed including both secondary and tertiary concentrates can be visually observed in Figures 3.1a to Figure 3.2b. Orange bars indicate samples that met or

exceeded the LOQ (≥3 GC per PCR reaction), whereas blue bars represent samples that were below the LOQ, but indicated the positive presence of the target sequence. During this investigation, the range for Norovirus was from 0.02 to 19065 GC/L for river water (Figure 3.2a) and from 0.02 to 272 GC/L for GW (Figure 3.1a). For Enterovirus the range observed was from

0.28 to 773 GC/L for river water (Figure 3.2b) and from 0.004 to 0.27 GC/L for GW (Figure

3.1b).

Closer analysis revealed the range for Norovirus and Enterovirus observed from secondary river water concentrates was from 0.0013 to 19065 GC/L and 0.64 to 773 GC/L, respectively. The range observed for Norovirus and Enterovirus from tertiary river water concentrates was between 67 and 738 GC/L and from 0 to 0.277 GC/L, respectively.

Norovirus concentrations between 0.065 and 44 GC/L were observed from GW secondary concentrate samples, and between 0.016 and 272 GC/L for tertiary concentrate samples.

Enterovirus concentrations between 0.032 and 0.105 GC/L were observed from the secondary concentrate samples and concentrations between 0.004 and 0.27 GC/L were seen in the tertiary concentrate samples.

Figure 3.1a

Figure 3.1b

Figures 3.1a and 3.1b Human Norovirus GII and human Enterovirus mean counts derived from duplicate tests for 21 untreated (raw) ground water source samples. Orange columns indicate samples positive for human Norovirus GII or human Enterovirus (Above LOQ ≥3 genomic copies per reaction). Blue columns indicate detections of human Norovirus GII or human Enterovirus below LOQ. Sample dates with no bars represent non-detects. Note, the Y-axis scale differs for each Figure. 56

Figure 3.2a

Figure 3.2b

Figures 3.2.a to 3.2b Human Norovirus GII and human Enterovirus mean counts derived from duplicate tests for 22 untreated (raw) river water source samples. Orange columns indicate samples positive for human Norovirus GII or human Enterovirus (Above LOQ, ≥3 genomic copies per reaction). Blue columns and numbers indicate detections of human Norovirus GII or human Enterovirus below LOQ. Sample dates with no bars represent non-detects. Note, the Y- axis scale differs for each Figure.

Table 3.1 illustrates the number of samples positive (detectable and or quantifiable) for human

Norovirus and human Enterovirus viral particles from the 21 GW and 22 river water source water samples within each season over a 16-month period. Human Norovirus was detected in both ground water and river water samples year-round when all positive detects were included in the analysis. This result is in agreement with the USA CDC statement that Norovirus infections occur year-round (CDC, 2017). Human Enterovirus was detected in raw groundwater samples in the spring summer and fall, whereas Enterovirus was detected in raw river water predominately in the winter (Table 3.1); this agrees with the observations of Fong and Lipp

(2005) from raw sewage and final wastewater effluents. The US CDC reported patterns for outbreaks of non-polio Enterovirus typically in the summer and fall (CDC, 2017).

Table 3.1 Samples positive for human Norovirus GII and human Enterovirus types as determined by qPCR listed by source water type and season. EV represents human Enterovirus; ND represents non detects and NoVGII represents human Norovirus GII Water Positives Positives Season 2015 Season 2016 Source Norovirus Enterovirus Winter Spring Summer Fall Winter Spring GII River 9 out of 22 4 out of 22 1 NoV 1NoV 2 NoV 2 NoV 3 NoV water- GII; GII GII GII GII; ND secondary 1EV 3 EV 41% 18% River 1NoV 2 NoV water- 3 out of 22 1 out of 22 ND GII GII ND ND ND tertiary 1 EV 14% 5% Ground 1NoV 1 NoV 2 NoV 1 EV Water - 4 out of 21 4 out of 21 GII GII; GII; ND ND secondary 2EV 1 EV 19% 19% Ground 1 NoV 2 NoV 3 NoV 1 NoV Water - 7 out of 21 5 out of 21 ND GII; 2 GII; 2 GII; 1 GII ND tertiary EV EV EV 33% 29%

3.4 Environmental trends:

River flow and precipitation impacts on Norovirus and Enterovirus detections were investigated by analyzing the presence of viral particles in relation to precipitation events (including snow melt events in the winter) and river flows. In addition to baseline sampling on monthly or biweekly schedules, we took additional samples during high risk periods; including rainfall events exceeding 15 mm within 48 h and snow-melt events that resulted in increased flow and surface water levels. Daily monitoring of weather on Environment Canada’s and Grand River

Conservation Authority’s website for precipitation and flow data, respectively, facilitated targeted sample collection. According to Environment Canada, there were 14 rain events that occurred between January 1, 2015 and April 30, 2016 in the study area that were ≥ 15 mm (ranging from

15.1 mm to 39.8 mm) within a 24 h period, six (6) of which occurred during the initial “routine monthly sampling” regime and were missed. For the remainder of the study, six (6) precipitation events were sampled (two were not), but viral particles were only detected by PCR assay for three (3) of the events. Two melt events were observed, one on January 11, 2016 and the second between March 10th and 12th 2016, whereby air temperatures were noted above seasonal that resulted in an increase in river flows greater than 100 m3/s. Both melt events were sampled, but viral particles (Norovirus) were detected only in the January event.

Occurrence of virus particles (GC/L) for human Norovirus GII and human Enterovirus from both

GW and river water samples with precipitation events and river flow can be visually observed in

Figure 3.3 to Figure 3.4b respectively. Upon examination of Figures 3.3 and Figures 3.4, an association of increased viral particle numbers (GC/L) with increased flow and precipitation events as hypothesized is not apparent for the river samples. For this investigation viral particles observed at the groundwater site were not hypothesized to increase with precipitation events and elevated groundwater level. This is because particle travel times were not known for

this sampling site. Further, level and flow monitors were not installed at this sampling site to monitor and record this information.

Figure 3.3a

Figure 3.3b

Figure 3.3a to 3.3b. Time series plot for January 2015 through to April 2016 of human Norovirus GII and human Enterovirus mean counts derived from duplicate tests for 21 untreated (raw) ground water source samples. vs. Precipitation for GW sites collected from the Government of Canada, Department of Environment rain gauge situated in Kitchener/Waterloo Ontario. Note, the secondary Y-axis scale differs for each Figure.

Figure 3.4a

Figure 3.4b

Figure 3.4a to 3.4b. Time series plot for January 2015 through to April 2016 of human Norovirus GII and human Enterovirus mean counts derived from duplicate tests for 22 untreated (raw) river water source samples vs. Precipitation for GW sites collected from the Government of Canada, Department of Environment rain gauge situated in Kitchener/Waterloo Ontario. Note, the secondary Y-axis scale differs for each Figure.

Figure 3.5 Time series plot for January 2015 through to April 2016 of river flows (daily mean (m3/s) collected from the Grand River Conservation Authority’s website situated in Kitchener/Waterloo Ontario and human Norovirus GII and human Enterovirus mean counts derived from duplicate tests for 22 untreated (raw) river water source samples. Data points include all giving a positive detection, i.e. data includes those below the LOQ.

3.5 Community prevalence and outbreak surveillance:

Wastewater from the wastewater treatment plant within the study area was sampled to confirm virus presence within the community during the winter and fall of 2015 and 2016. Norovirus was detected in all samples (January and September 2015; February 2016), whereas, Enterovirus was detected in only one sample (Fall 2015) (Table 3.2). The Region of Waterloo reported 44 enteric outbreaks between September 1, 2015 and April 30, 2016, whereby 50 % of these occurred in child care facilities. Norovirus was the most frequently detected agent in these outbreaks which were observed to peak between January and March (Region of Waterloo,

2016).

Table 3.2 Wastewater treatment grab samples positive for human Norovirus GII and human Enterovirus types as determined by qPCR.

Sample Date Sample Type Norovirus (GC/L) Enterovirus (GC/L)

January 2015 wastewater within the 3.7 X 104 ND* UV treatment chamber

September 2015 wastewater within the 3.8 x 104 Present (below LOQ) UV treatment chamber

February 2016 raw sewage influent 1.1 x 104 ND*

* ND represents non-detects

3.6 Statistical correlations:

The numbers of virus particles present in water was estimated by RT-qPCR based on comparison to 10-fold serially diluted standard plasmids containing the target gene. All statistical analysis was performed on the back-calculated values from the qPCR results expressed as GC/L filtered water (Appendix 5).

To test for correlations between virus counts and precipitation, flow, turbidity, temperature, conductivity, and E. coli and total coliform, the Pearson’s correlation test was initially attempted.

However the number of positive qPCR detects within the quantification range (≥3 GC) was as low as n=2 (Enterovirus presence in river water). These low n values generated errors within the operation of the probability test equation as a minimum of 3 matched pairs are required for the operation to generate an output value, and the Pearson’s formula generated errors when non-detects were entered as zero. As a result a logistic regression was used to evaluate the data set. Using a logistic regression enabled the evaluation of all sampling events by converting positive qPCR detects to one (1) and non-detects to zero (0), thus producing a binomial data set. The logistic analysis approach facilitated the calculation of the probability of the viral

particle being associated with the physical and chemical parameters investigated (p) and probability of viral particle presence or absence (odds ratio). The results of the logistic regression can be found in Tables 3.2 and 3.3 for river water and GW, respectively, where P <

0.05 has been selected as statistically significant.

When logistic regression was used to determine correlations between virus counts observed at the river water and GW sites and physical, chemical and microbiological factors, no statistically significant correlations were observed (p <0.05) with the exception of, a slight positive correlation which was observed between Norovirus at the river water site and water temperature

(p = 0.007). Moderate evidence suggests increased Norovirus GII nucleic material was detected at lower water temperatures. Greer et al. (2009) reported an association between Norovirus occurrence in the community with decreasing Lake Ontario temperatures, as such it was prudent to perform comparative analysis on the Grand River samples to see if temperature was a potential factor for the determination of Norovirus GII presence.

Human Norovirus GII counts plotted with river water temperature can be seen in Figure 3.10 below. Positive correlation values (p <0.05) were not reported for viral particle detection when compared to physical, chemical or microbiological parameters when logistic regression analysis was applied to the data set. Therefore, concluding the associations are not statistically significant even though a few odds ratio values reported are greater than the value of 1 in the case of precipitation, flow, indicator bacteria, and conductivity for Norovirus and Enterovirus

(Tables 3.2 and 3.3, higher odds of viral particle presence associated with the parameter increasing or decreasing).

Table 3.3 Statistical values of probability using logistic regression comparing virus counts (GC/L) between river water (RW) source and precipitation, river flow and river level, turbidity, temperature, conductivity; and microbiological counts. All results were used.

RIVER NOROVIRUS Sampl Precipitatio River River Turbidity Temp Conductivit E. coli Total WATER e type n Flow Level (NTU) (0C) y (µS) (CFU/100mL coliform RAW: ) (CFU/100mL ALL ) DATA Probability (p) RW 0.930 0.764 0.763 0.291 .007 0.485 0.378 0.318 Odds ratio RW 1.005 0.997 0.499 0.951 1.169 0.998 1.001 1.000 ENTEROVIRUS Probability RW 0.613 0.865 0.637 0.962 0.499 0.544 0.867 0.480 (p<0.05)) Odds ratio RW 0.966 1.002 3.346 0.998 0.946 1.002 1.000 1.000

Table 3.4 Statistical values of probability using logistic regression comparing virus counts (GC/L) between GW source and precipitation, river flow and river level, turbidity; and microbiological counts. All results were used

GROUND NOROVIRUS Sample Precipitatio River River Turbidity E. coli Total WATER type n Flow Level (NTU) (CFU/10 coliform RAW: ALL 0mL) (CFU/100mL DATA ) Probability (p) RW 0.930 0.764 0.763 0.291 0.378 0.318 Odds ratio RW 1.005 0.997 0.499 0.951 1.001 1.000 ENTEROVIRUS Probability RW 0.613 0.865 0.637 0.962 0.867 0.480 (p<0.05) Odds ratio RW 0.966 1.002 3.346 0.998 1.000 1.000

Figure 3.6 Plot of Norovirus count vs. Temperature data for January 2015 through to April 2016 of Ambient air temperature collected from the Government of Canada, Department of Environment rain gauge situated in Kitchener/Waterloo Ontario and human Norovirus GII mean counts derived from duplicate tests for 22 untreated (raw) river water source samples.

Chapter 4: Discussion

This investigation examined the presence of human Norovirus and human Enterovirus in

Ontario at two source water sites, a surface water site and a ground water site.

Monitoring for enteric viruses in Ontario, sources of drinking water provides data for quantitative microbial risk analyses (QMRA), for the purpose of assessing risk and to inform decisions on the approach and implementation of prevention, control, and treatment strategies for effective water treatment operations. Enteric virus monitoring, for at minimum a year that includes seasonal variability and short term incidental or peak moments (heavy rainfall) is necessary to achieve a representative quantification of the number of Enterovirus and Norovirus present in source water for input into QMRA platforms (Schijven et al. 2011).

It was hypothesized that raw source waters would contain human Norovirus GII and human

Enterovirus, which would be detectable in concentrated untreated river water samples and correlate with higher turbidity, precipitation events and river flow measurements. Further, human

Norovirus GII and human Enterovirus detection would correspond with late fall to early spring, and mid-summer to early fall, respectively, mirroring monitored and reported community associated illness and outbreaks.

It was also predicted that human Norovirus GII and human Enterovirus may be detectable in concentrated untreated ground water samples as a result of virus particles entering the aquifer, as this groundwater source is considered to be groundwater under the influence of surface water (GUDI). Thus, the groundwater source can be impacted by anthropogenic activities

(effluent from large urban wastewater treatment facilities, and rural septic systems); and that occurrence would follow similar seasonality and correlate with precipitation events and other physical-chemical parameters.

Using a method based on USEPA1615, RT-qPCR was employed in this study to semi- quantitatively determine the presence of human Norovirus GII and human Enterovirus in raw river water and raw groundwater. Wastewater samples obtained from a wastewater treatment facility from within the study area during the fall of 2015 and winters of 2015 and 2016 detected the presence of Norovirus GII and Enterovirus, thus verifying their presence within the community and study area; and likelihood of these pathogens being present in downstream receiving river water and ground water.

Sampling was conducted over a sixteen month timeframe whereby baseline and event-based sampling generated a sample size of n=22 for river water and n=21 for GW samples. Overall, 5

– 41% of samples were found to be positive for human Norovirus and/or Enterovirus, thus the majority of samples had non-detects from both sampling sites (Table 3.1)

4.1 Recovery of MS2 as a surrogate using USEPA 1615

MS2 was selected as a representative enteric RNA virus surrogate of Norovirus and Enterovirus for the purpose of determining enteric virus recoveries from both river water and GW

(environmental waters) in this investigation. Bae et al. (2008) reported no statistical significance in nucleic acid reductions in environmental water at 4°C, and removal rates during drinking water treatment and disinfection processes between MS2 and NV; and MS2 also exhibited seasonal variation with associated disease incidence. In the laboratory, MS2 virus recoveries of

GW were determined to be 5 % for undiluted secondary concentrated samples and 38% for undiluted tertiary concentrated samples. This is lower than the mean recovery of 44% for MS2 from groundwater as reported by Bennett et al. (2010). In this investigation mean recoveries of

30% were observed for secondary concentrate samples from river water sources and 3% for tertiary concentrates. A range of MS2 recoveries have been reported in the published literature

and are dependent on the water (deionized, sea water, surface water, or tap water) and filter type (Nanoceram, IMDS, and flat cellulose) used in the experiment (Lambertini et al. 2008).

Mull et al. (2012) reported MS2 mean recoveries from hollow micro fiber filters of 66% from 40 L of lake water seeded with up to 4.7 x 10 5 PFU. Recovery from river water in this study is similar to the one reported by Polaczyk et al. (2007): 32% mean recovery from 20 L samples of filtered tap water (IMDS cartridge filter) seeded with 104 MS2.

4.2 Prevalence of Norovirus and Enterovirus

In this investigation the concentration of Norovirus GII and Enterovirus detected in untreated

GW ranged between 0.065 and 273 GC/L, and 0.005 to 0.27 GC/L, respectively. These values are similar to those reported by others who used molecular methods to detect Enterovirus and

Norovirus in groundwater. Lambertini et al. (2012), detected Enterovirus at mean concentrations of 0.9 GC/L, and detected Norovirus GI at 2.06 GC/L in untreated well water when analyzed by RT-PCR. Further, the United States reported 31.5% of drinking water wells positive for Enterovirus when analyzed by RT-PCR (Borchardt et al. 2004).

Norovirus GII and Enterovirus detected in untreated river water were observed to be between

0.017 and 19065 GC/L and 0.23 and 773 GC/L, respectively, in this investigation. Lodder et al.

(2010) reported detections of Enterovirus averaging 0.0052 - 2.4 PFU/L, and Norovirus averaging 0 to 171 PCR-detectable units (PDU)/L of surface water. Lazic et al. (2015) reported

Norovirus GI in river waters from Serbia between 0 – 960 PDU/L. Norovirus GII concentrations detected in this study are similar, but acknowledge Enterovirus concentrations are higher, likely as a result of an overestimation effect of non-viable virus particle detection by PCR as compared to the detection of Enterovirus by culture methods employed by Lodder et al. (2010).

Prevost et al. (2016) reported median concentrations of Enterovirus and Norovirus at 3 GC/L

(median) and 25 GC/L (median), respectively, in river waters from France.

The majority of samples analyzed in this investigation generated results less than the limit of quantification (LOQ≥ 3 GC/PCR reaction). Sample analysis employing technical triplicates is the approach most often cited for qPCR assay (USEPA, 1615), however this study consumed a vast quantity of cDNA in performing inhibition testing on each sample for both Norovirus GII and

Enterovirus primer-probe sets and therefore technical duplicates for each sample was the approach used in the final qPCR assay. This approach however, did record incidences whereby one sample detected the presence of nucleic acid and the second technical replicate did not. This was the case for river water field samples and the Enterovirus assay. Detections of Enterovirus were observed to be below the LOQ and for every sample, only one of the duplicate tests detected the presence of Enterovirus at these low concentrations. Enteric viruses have been reported to aggregate (Cashdollar et al. 2013; and Teunis et al. 2008). Low viral particle numbers and viral particle aggregation may create a situation where the number of viral particles reported for each aliquot differs. A study to determine the optimal number of samples required to detect the positive target within the volume of sample should be determined to avoid under reporting or reporting the absence of viral particles in a sample.

4.3 Seasonal Occurrence of Norovirus and Enterovirus

Seasonality of viral infections of humans by Norovirus and Enterovirus is a long-recognized

(Fernandez et al. 2012; and Greer et al. 2009). A better understanding of the relationship between seasonality and environmental factors could provide more accurate information to water treatment operations for QMRA, which in turn could reflect responsive changes in water

treatment processes during predictable periods of correlation between disease incidence and seasonal exposures.

Seasonal patterns were observed for Enterovirus in groundwater, whereas Norovirus GII was detected all year long. Illness attributable to Norovirus typically increases throughout the fall before peaking in January and then decreases in the spring with reduced activity during the summer (Maunula et al. 2005). In this study samples were collected across all seasons, with

Norovirus GII being detected all year long in both the river and groundwater sources. Norovirus

GII concentrations in this study were observed to be higher in river water samples for the winter months of January and March ranging from 749 – 1546 GC/L, which was also reported by

Westerall et al. (2006), where she observed >100 pdu/L of human Norovirus during the winter seasons only. In this study, groundwater concentrations of Norovirus GII however, were observed higher in late winter (March) and early fall (September), and at lower concentrations as compared to river water samples 55.7 – 272 GC/L (Appendix 2). Norovirus concentrations were also observed to be higher in ground water for the late summer and early autumn months of September and October in 2015 where elevated flow and rain events were concurrently recorded on the day of sampling. These results are possibly indicative of an elevation of human

Norovirus GII illness circulating within the municipality prior to sampling. Waterloo Region reported Norovirus activity from one institutional outbreak for the month of October, 2015

(Region of Waterloo, 2016). Elevated concentrations could also be reflective of increased recreational use of the upstream river water source that influences this GW in the summer months (Lodder and de Roda Husman, 2005). The presence of Norovirus in groundwater for

October 2015 is supported by the positive detection of Norovirus GII from the untreated wastewater sample for September 2015 where 13440 GC/L of human Norovirus GII was detected in the influent and 33790 GC/L was detected in the wastewater UV treated effluent going out to the receiving Grand River source within the study area.

Enterovirus outbreaks can occur throughout the year depending on the type of Enterovirus, but in temperate climates, Enterovirus infections have been observed mostly during late summer months (Moore et al. 1982; Sedmak et al. 2003). Similarly in this study, Enterovirus was detected in the spring, summer and fall for the groundwater samples (≤ 1 GC/L), and summer months from river water samples (≤ 1 GC/L) (Appendix 4). Enterovirus was detected in the winter months, but concentrations were below 1 GC/L. The highest concentration of Enterovirus was observed from river water samples taken in the early spring for both 2015 (5.9 GC/L) and

2016 (773 GC/L). This is in agreement with Costan-Longares et al. (2008), who reported the isolation of Enterovirus, Coxsackievirus B4 from river water samples (75% samples positive, at a geometric mean of 0.4 PFU/L) and wastewater in the early summer in Spain.

4.4 Meteorological, Physical and Chemical Interactions

We hypothesized that the detection of human Norovirus GII and human Enterovirus in the raw river water would increase as precipitation, flow and turbidity increased. Quantifiable concentrations of Norovirus GII were reported in 2015 for two river samples taken during rain events in mid-June and July of 2015 (5531 – 19065 GC/L), in which, daily average precipitation was 18mm and 20 mm (daily average) respectively, and river flows were elevated at 103 and 20

(m3/s) respectively. Yet over the 16 month period, no correlation between viral counts and precipitation, or river flow was observed (Table 3.2).

Norovirus GII concentrations were observed to be higher in ground water for the late summer and early autumn months of September and October in 2015. These two sampling dates were also associated with elevated river flow and rain events (16mm and 18 mm daily average precipitation respectively), which may suggest a hydrogeological connection in the absence of

hydrogeological data for this site, however, over the 16 month period, no correlation between precipitation, or river flow was observed (Table 3.3).

More frequent sampling or a multi-year study may be necessary to determine if elevated concentrations in water are as a result of precipitation or flow events or recreational use, as a low number of significant precipitation events were observed within the study area between

2015 and spring of 2016. The GRCA reported 2016 as a dry year, and reported 2015 also as having had dryer than normal conditions in the summer months (GRCA, 2016). With few precipitation events there is an opportunity for future sampling programs to review optimal sampling times. Testing of samples collected during different times within an event is needed to determine the optimal time of sampling to improve detection and sensitivities of the sampling protocol. In this study samples were taken within 48 h of the peak of the precipitation or melt event. It is possible that the non-detects reported in this study indicate viral particles were missed using a post 48 h event for river water sampling. Hata et al. (2014) reported an increase in Norovirus GII and Enterovirus by RT-qPCR during rainfall events (samples taken during the rain and up to 24 h after the rain). Perhaps the post 48 h point in this study was too early for

GW sampling. Knowing the time of travel for GW sites would be necessary to inform optimal sampling times relative to precipitation and melt events to capture transport of the highest possible density of viral particles from the shallow sub surface aquifer to the GW site.

It is worth noting that in 2016, Norovirus GII concentrations were observed to be higher in the river water for the winter months of January and March that were not associated, nor correlated with elevated flow or rain events. However, it was noted through local news reports that the

January sample was taken during a week in which suspected outbreaks of Norovirus GII within the community were on the rise. The Region of Waterloo for January of 2016 reported 8 institutional outbreaks, but community surveillance programs were not in place for the reporting of individual illnesses for Norovirus and Enterovirus. In this study a raw sewage influent sample

collected on February 4, 2016, during this rise in community ‘Norovirus-like symptoms reporting’, was determined to contain 10,900 GC/L of human Norovirus GII.

Results from this study indicate no relationship between the presence of Norovirus GII and

Enterovirus in river water with conductivity (Table 3.2). However, a slight correlation was found between Norovirus GII occurrence and colder water temperatures for river water samples when logistic regression was applied using all test results. This is in agreement with Greer et al.

(2009) who reported an association between Norovirus occurrence in the community with decreasing Lake Ontario temperatures upon analyzing Ontario gastroenteritis outbreak data and employing regression models. No correlation was observed for Enterovirus from river water samples in this investigation.

4.5 PCR detection and determination of infectivity of Norovirus and

Enterovirus

This study employed qPCR method of detection, which does not permit the discrimination between infectious and non-infectious virus particles. Results are based on PCR detection of the specific target nucleic acid sequences for Norovirus GII and Enterovirus. However, the results do not indicate infectivity. Infectivity of samples collected in this study could be demonstrated in a future study employing animal models for a feed investigation, in the absence of a validated cell culture method, for Norovirus (Atmar and Estes, 2001); and infectivity of

Enterovirus positive samples could be demonstrated through subsequent determination of cytopathic effects in cell culture using A549 and BGM cell lines for example (Lee et al. 2004).

To our knowledge, no published studies had reported on method comparison and recovery for human Norovirus GII from PCR and culture based methods to facilitate discussion on the potential for over estimation of Norovirus detections in GW and river waters in this study.

Culture based methods for human Norovirus from environmental samples have yet to be developed, however some studies showed that human Norovirus could be cultured in the lab for infectivity demonstration (Subba-Reddy et al. 2011; Wobus et al. 2004). The lack of correlation between the detection and quantification of viral genome sequences by PCR and cell culture is well known and has been reported previously for enteric viruses such as human Adenovirus, feline Calicivirus, and murine Norovirus (Baert et al. 2008; Choi, and Jiang, 2005; Enriquez et al.

1993; Gassilloud et al. 2003). These studies report higher recoveries for PCR detection methods than those employing culture based and confirm no single cell line works to culture all enteric virus types. For instance, Puig et al. (1994), reported PCR detection of ≤ 10 particles of human Enterovirus in their study translating to 100 to 1000 times higher than the sensitivity of cell culture using the standard BGM cell line.

Discerning non-infectious Norovirus and Enterovirus from infectious virus was outside the scope of this investigation. Recent studies have suggested treating samples before nucleic acid extraction with intercalating dyes such as ethidium monoazide (EMA) or propidium monoazide

(PMA) so that viral particles with damaged capsids, presumed inactivated and non-infectious, are unable to be amplified by PCR (McLellan et al. 2016). This area of research is emerging and there is a need to perform studies using environmental samples from within the study area to provide inputs into QMRA on the number of infectious viral particles. Archived samples from this investigation could be used in future investigations to ascertain infectious viral particle concentrations to further inform risk models and managers of treatment systems.

4.6 Bacterial Indicators as surrogates for Norovirus and Enterovirus

Data on two microbiological indicators, used internationally for decades as determinants of water quality, indicative of environmental contamination (total coliform) or faecal pollution (E.

coli) were also obtained on the sampling dates within this study. Upon review it was observed that, all GW samples were negative for total coliforms and E. coli. In contrast, 19-33% of samples were positive by RT-PCR for Norovirus GII and Enterovirus (Appendix 4). By this comparison, indicator bacteria were not significantly correlated with virus occurrence in groundwater, as also demonstrated previously by other investigators (Abbaszadegan et al.

2003; Borchardt et al. 2003; Gerba et al. 1984; Jiang et al. 2001). As such, they may not be good indicators of viral presence and therefore overall water sanitation or quality of this ground water source. The ground water source in this study was identified as ground water under the direct influence of surface water (upstream river) based on vicinity to the aquifer, which is shallow and directly beneath the river upstream. As such it seemed reasonable to hypothesize the presence of total coliforms and/or E. coli during or subsequent to precipitation and melt events. The challenge, however, is that the travel time for this site is unknown as no tracer studies for this site had been performed and no estimated times were presented in GW site engineering reports. Perhaps the sample size of 100 mL for the total coliform and E. coli microbiological culture based method used was not able to detect their presence. Sample volumes collected (30 to 1500 L for groundwater samples) to detect the nucleic acid material of

Norovirus GII and Enterovirus were hundreds to tens of thousands times greater than those used to detect bacterial targets (100 mL sample). As such, future investigation could test archived samples from this study (comparable sample volumes) for the presence of nucleic sequences specific to the bacterial indicators. For example, for E. coli targeting the uidA and uidR genes (Deshmukh et al. 2016); and specific types within the indicator total coliform grouping (e.g., Enterococcus spp. 23S rDNA) could be used for detection. River water and GW faecal indicator results can be seen in Appendix 4. E. coli and total coliform counts were reported for the river water samples, but no correlation with Norovirus GII or Enterovirus was found (Table 3.2). This is in agreement with published studies in which raw water samples of

100 mL, or 1 L volumes were collected and analyzed (Abbaszadegan et al. 2003; Borchardt et al. 2003; and Jiang et al. 2001).

5.0 Future Perspectives

This study served as an essential first step in advancing our laboratory’s approach to source water characterization of human pathogenic enteric viruses. This investigation was comprised of two different drinking water sources that were routinely sampled over 1.25 years to monitor human Norovirus GII and Enteroviruses. Future work should review and include epidemiologically important and emerging enteric viruses within the watershed, to obtain a more detailed picture of viral contamination in surface and source waters. Examples may include

Rotavirus (2011 Ontario adopted Rotavirus infant vaccination program), Adenovirus, Astrovirus and specific Enterovirus types (Echovirus). Additionally the coordination of public health surveillance for reporting on individual illness rates and identification or sequencing of human

Enterovirus types would have assisted in the design of more specific Enterovirus primer probe sets.

Shifting perspective to a watershed level would take into account the community endemic and fluctuating illness rates, to facilitate the selection of a broad range of specific primer and probe sets for the broad grouping of types within the Enterovirus group. Sampling and monitoring of institutional wastewaters including hospital discharge pipes would identify enteric virus types circulating within the community that travel downstream to become diluted at receiving wastewater treatment facilities that could make their way into receiving source waters. Source water sampling at locations between the wastewater treatment outfall and downstream drinking water treatment intakes could inform viral particle presence.

The characterization of source waters intended for drinking water is necessary for establishing effective and optimized treatment systems. Establishing a source water characterization protocol for viral pathogens requires research, research into optimal sampling volumes, sampling timing and processing chemistries. Characterization of source waters should be approached in a customized fashion whereby each source water study is designed to determine the presence and quantity of applicable viral particles of concern, which then can be provided to operational managers for input into QMRA tools to facilitate decision making processes.

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APPENDICES

Appendix 1: Sampling Record

YEAR 2014 2015

DATE Dec Dec Dec Jan Jan Jan Jan Feb Feb Feb 14- 21- 28-3 4-10 11-17 18-24 25-31 1-7 8-14 15-21 20 27

GW X X

RR X X

WW X RAW

YEAR 2015 cont’d

DATE Feb March March March March March April April April April 22-28 1-7 8-14 15-21 22-28 29-4 5-11 12-18 19-25 26-2

GW X X X(spike)

RR X X X(spike)

WW RAW

YEAR 2015 cont’d

DATE May May May May June June June June June July 3-9 10-16 17-23 24-30 31-6 7-13 14-20 21-27 28-4 5-11

GW X X

RR X X

WW RAW

YEAR 2015 cont’d

DATE July July July Aug Aug Aug Aug August Sept Sept Sept Sept Oct Oct Oct 12- 19- 26- 2-8 9- 16- 23- 30-5 6-12 13- 20- 27-3 4- 11- 18- 18 25 1 15 22 29 19 26 10 17 24

GW X X X X X

RR X X X X X

WW RAW

YEAR 2015 cont’d

DATE Oct Nov Nov Nov Nov Nov Dec Dec Dec 25-31 1-7 8-14 15-21 22-28 29-5 6-12 13-19 20-26

GW X X X

RR X X X

WW RAW

YEAR 2016

DATE Jan Jan Jan Jan Feb Feb Feb Feb March March March March 1-9 10-16 17- 24- 31-6 7-13 14-20 21-27 28-5 6-12 23 30

GW X X X X X X X

RR X X X X X X X

WW X X RAW

YEAR 2016 cont’d

DATE April April Apr il

GW X X

RR X x

WW RAW

Appendix 2: Tables of study data

Table 2a Study data for all processed samples from Ground Water (GW) source

Sample ID Sample Turbidity Temp E. coli Total coliform Norovirus Enterovirus type (NTU) (ºC) (CFU/100mL) (CFU/100mL) (GC/L) (GC/L)

January 13, 2015 GW 0.12 N/A 0.00E+00 0.00E+00 0.207 ND

February 17, 2015 GW 0.18 N/A 0.00E+00 0.00E+00 lab error Lab error

March 5, 2017 GW 0.1 N/A 0.00E+00 0.00E+00 ND ND

March 18, 2015 GW N/A N/A N/A N/A ND ND

April 14, 2015 GW N/A N/A N/A N/A ND 0.058

May 8, 2015 GW N/A N/A N/A N/A ND 0.015

June 12, 2015 GW N/A N/A N/A N/A 0.065 0.272

July 17, 2015 GW N/A N/A N/A N/A 8.720 0.064

Sept. 15, 2015 GW N/A N/A N/A N/A 272.293 0.033

Sept. 30, 2015 GW 0.19 N/A 0.00E+00 0.00E+00 ND ND

Oct. 17, 2015 GW 0.15 N/A 0.00E+00 0.00E+00 55.704 ND

Oct. 29, 2015 GW 0.15 N/A 0.00E+00 0.00E+00 0.423 0.105

Nov. 14, 2015 GW 0.16 N/A 0.00E+00 0.00E+00 1.392 ND

Nov. 28, 2015 GW 0.16 N/A 0.00E+00 0.00E+00 ND 0.005

Jan. 2, 2016 GW N/A N/A 0.00E+00 0.00E+00 ND ND

Jan 13, 2016 GW 0.13 N/A 0.00E+00 0.00E+00 ND ND

Jan. 30, 2016 GW N/A N/A 0.00E+00 0.00E+00 ND ND

Feb. 4, 2016 GW 0.1 N/A 0.00E+00 0.00E+00 ND ND

Feb. 21, 2016 GW 0.1 N/A 0.00E+00 0.00E+00 53.43 ND

March 12, 2016 GW 0.25 N/A 0.00E+00 0.00E+00 ND ND

April 3, 2016 GW 0.27 N/A 0.00E+00 0.00E+00 ND ND

April 17, 2016 GW 0.27 N/A N/A N/A ND ND

Table 2b Study data for all processed samples from River Water (RW) source

Sample ID Sampl Turbidity Temp Conductivity E. coli Total coliform Norovirus Enterovirus e type (NTU) (ºC) (µS) (CFU/100mL) (CFU/100mL) (GC/L) (GC/L)

January 13, RW 1.71 3.1607 816.80 1.60E+01 200 ND ND 2015

February 17, RW 1.57 2.163 840.06 9.00E+00 100 ND ND 2015

March 5, 2017 RW N/A N/A N/A N/A N/A ND ND

March 18, RW 14.56 1.8743 608.54 1.00E+0 58,000 0.017 5.854 2015

April 14, 2015 RW 8.96 5.6595 680.19 1.50E+0 400 ND ND

May 8, 2015 RW 1.24 18.338 712.85 5.00E+0 200 ND ND

June 12, 2015 RW 3.10 15.681 569.80 9.90E+0 1000 19065.341 ND

July 17, 2015 RW 7.05 20.951 659.85 5.00E+01 1000 5531.494 0.278

Sept. 15, RW 4.68 18.223 524.78 1.50E+01 3000 0.009 ND 2015

Sept. 30, RW 6.00 18.291 445.23 5.00E+01 150 67.046 ND 2015

Oct. 17, 2015 RW 2.91 12.348 436.75 3.80E+01 200 ND ND

Oct. 29, 2015 RW 6.88 11.244 421.60 2.30E+01 500 0.001 ND

Nov. 14, 2015 RW 4.84 8.8482 591.92 2.40E+01 1500 ND ND

Nov. 28, 2015 RW 38.62 8.4428 574.34 9.90E+01 5000 ND ND

Jan. 2, 2016 RW 11.13 4.2712 588.87 2.40E+01 1000 1392.106 ND

Jan 13, 2016 RW 10.07 3.036 577.13 1.90E+03 9.10E+04 748.934 ND

Sample ID Sampl Turbidity Temp Conductivity E. coli Total coliform Norovirus Enterovirus e type (NTU) (ºC) (µS) (CFU/100mL) (CFU/100mL) (GC/L) (GC/L)

Jan. 30, 2016 RW 1.82 2.6341 762.17 5.00E+00 N/A ND 0.803

Feb. 4, 2016 RW 42.17 N/A 493.42 6.80E+02 7000 ND ND

Feb. 21, 2016 RW 20.22 3.659 601.57 1.10E+02 5000 ND 0.642

March 12, RW 7.17 5.7421 442.33 1.60E+01 2100 1546.437 773.219 2016

April 3, 2016 RW 18.00 5.8817 482.29 1.90E+01 1200 ND ND

April 17, 2016 RW 5.97 10.391 654.10 3.00E+00 100 ND ND

Appendix 3: Inhibition test results

Table 3a Inhibition data for all processed samples from Ground Water (GW) source

Sample spiked cDNA sample with Sample Norovirus type Norovirus Enterovirus spiked with Sample Source GII (secondary cDNA GII plasmid Enterovirus ID water plasmid concentrate dilution plasmid control (CT plasmid (CT type control (date) or tertiary (CT mean Mean) mean of (CT Mean) concentrate) of duplicates) duplicates ) January 13, 2015 GW secondary undiluted 34.6 35.00 25.80 29.90 GW secondary 1:2 34.6 34.50 25.80 26.70 GW secondary 1:4 34.6 34.00 25.80 25.60 GW secondary 1:8 34.6 34.90 25.80 25.40 GW tertiary undiluted 34.6 34.20 25.80 33.30 GW tertiary 1:2 34.6 34.40 25.80 26.30 GW tertiary 1:4 34.6 25.80 25.70 GW tertiary 1:8 34.6 25.80 25.70 Februar y 17, 2015 GW secondary undiluted lab error ND ND ND GW secondary 1:2 lab error ND ND ND GW secondary 1:4 lab error ND ND ND GW secondary 1:8 lab error ND ND ND GW tertiary undiluted lab error ND ND ND GW tertiary 1:2 lab error ND ND ND GW tertiary 1:4 lab error ND ND ND GW tertiary 1:8 lab error ND ND ND March 5, 2015 GW secondary undiluted 35.2 35.50 26.10 25.20 GW secondary 1:2 35.2 34.60 26.10 26.30 GW secondary 1:4 35.2 34.50 26.10 26.30 GW secondary 1:8 35.2 34.50 26.10 24.10 GW tertiary undiluted 35.2 35.00 26.10 32.80 GW tertiary 1:2 35.2 33.70 26.10 27.10 GW tertiary 1:4 35.2 34.90 26.10 26.30

Sample spiked cDNA sample with Sample Norovirus type Norovirus Enterovirus spiked with Sample Source GII (secondary cDNA GII plasmid Enterovirus ID water plasmid concentrate dilution plasmid control (CT plasmid (CT type control (date) or tertiary (CT mean Mean) mean of (CT Mean) concentrate) of duplicates) duplicates ) GW tertiary 1:8 35.2 34.20 26.10 26.00 March 18, 2015 GW secondary undiluted 35.2 35.20 26.10 32.60 GW secondary 1:2 35.2 35.60 26.10 28.80 GW secondary 1:4 35.2 34.90 26.10 26.20 GW secondary 1:8 35.2 34.90 26.10 26.20 GW tertiary undiluted 35.2 35.70 26.10 32.80 GW tertiary 1:2 35.2 35.50 26.10 27.10 GW tertiary 1:4 35.2 33.80 26.10 26.30 GW tertiary 1:8 35.2 34.60 26.10 26.00 April 14, 2015 GW secondary undiluted 34.30 35.30 23.60 30.80 GW secondary 1:2 34.30 34.00 23.60 24.40 GW secondary 1:4 34.30 34.20 23.60 23.90 GW secondary 1:8 34.30 34.20 23.60 23.60 GW tertiary undiluted 34.30 34.40 23.60 33.70 GW tertiary 1:2 34.30 34.10 23.60 24.80 GW tertiary 1:4 34.30 34.50 23.60 23.70 GW tertiary 1:8 34.30 33.90 23.60 23.60 May 8 2015 GW secondary undiluted 34.30 35.90 23.60 29.90 GW secondary 1:2 34.30 34.70 23.60 25.20 GW secondary 1:4 34.30 34.30 23.60 24.20 GW secondary 1:8 34.30 34.50 23.60 23.80 GW tertiary undiluted 34.30 35.10 23.60 31.60 GW tertiary 1:2 34.30 33.70 23.60 25.30 GW tertiary 1:4 34.30 34.20 23.60 23.90 GW tertiary 1:8 34.30 34.00 23.60 23.70 June 12 2015 GW secondary undiluted 34.30 34.90 23.60 31.10 GW secondary 1:2 34.30 31.50 23.60 25.50

100

Sample spiked cDNA sample with Sample Norovirus type Norovirus Enterovirus spiked with Sample Source GII (secondary cDNA GII plasmid Enterovirus ID water plasmid concentrate dilution plasmid control (CT plasmid (CT type control (date) or tertiary (CT mean Mean) mean of (CT Mean) concentrate) of duplicates) duplicates ) GW secondary 1:4 34.30 32.40 23.60 23.70 GW secondary 1:8 34.30 33.50 23.60 24.60 GW tertiary undiluted 34.30 32.40 23.60 29.30 GW tertiary 1:2 34.30 32.40 23.60 25.60 GW tertiary 1:4 34.30 32.80 23.60 28.70 GW tertiary 1:8 34.30 33.50 23.60 23.50 July 17, 2015 GW secondary undiluted 34.30 35.30 23.60 29.40 GW secondary 1:2 34.30 34.20 23.60 25.20 GW secondary 1:4 34.30 34.80 23.60 23.80 GW secondary 1:8 34.30 33.80 23.60 23.60 GW tertiary undiluted 34.30 35.00 23.60 31.60 GW tertiary 1:2 34.30 34.10 23.60 25.00 GW tertiary 1:4 34.30 33.70 23.60 23.70 GW tertiary 1:8 34.30 34.10 23.60 23.50 Sept 15 2015 GW secondary undiluted 34.30 35.70 23.60 27.30 GW secondary 1:2 34.30 34.80 23.60 25.10 GW secondary 1:4 34.30 34.80 23.60 23.70 GW secondary 1:8 34.30 34.40 23.60 23.60 GW tertiary undiluted 34.30 34.60 23.60 28.20 GW tertiary 1:2 34.30 33.50 23.60 24.20 GW tertiary 1:4 34.30 33.30 23.60 23.60 GW tertiary 1:8 34.30 34.00 23.60 23.40 Sept 30 2015 GW secondary undiluted 34.55 36.10 25.81 35.70 GW secondary 1:2 34.55 34.50 25.81 26.99 GW secondary 1:4 34.55 34.60 25.81 25.70 GW secondary 1:8 34.55 34.30 25.81 25.70 GW tertiary undiluted 34.55 34.30 25.81 34.60

101

Sample spiked cDNA sample with Sample Norovirus type Norovirus Enterovirus spiked with Sample Source GII (secondary cDNA GII plasmid Enterovirus ID water plasmid concentrate dilution plasmid control (CT plasmid (CT type control (date) or tertiary (CT mean Mean) mean of (CT Mean) concentrate) of duplicates) duplicates ) GW tertiary 1:2 34.55 34.00 25.81 26.82 GW tertiary 1:4 34.55 34.60 25.81 25.88 GW tertiary 1:8 34.55 34.70 25.81 25.80 Oct 17 2015 GW secondary undiluted 34.55 35.60 25.81 33.75 GW secondary 1:2 34.55 34.30 25.81 27.50 GW secondary 1:4 34.55 34.40 25.81 25.87 GW secondary 1:8 34.55 34.40 25.81 25.60 GW tertiary undiluted 34.55 35.30 25.81 35.70 GW tertiary 1:2 34.55 34.70 25.81 27.45 GW tertiary 1:4 34.55 34.40 25.81 25.75 GW tertiary 1:8 34.55 34.10 25.81 25.60 Oct 29 2015 GW secondary undiluted 34.55 34.20 25.81 34.10 GW secondary 1:2 34.55 34.80 25.81 29.30 GW secondary 1:4 34.55 34.30 25.81 25.80 GW secondary 1:8 34.55 34.50 25.81 25.70 GW tertiary undiluted 34.55 34.10 25.81 UND GW tertiary 1:2 34.55 34.20 25.81 27.70 GW tertiary 1:4 34.55 34.20 25.81 25.70 GW tertiary 1:8 34.55 34.60 25.81 25.62 Novemb er 14 2015 GW secondary undiluted 34.55 35.60 25.81 34.10 GW secondary 1:2 34.55 34.20 25.81 27.70 GW secondary 1:4 34.55 34.40 25.81 25.99 GW secondary 1:8 34.55 34.40 25.81 25.70 GW tertiary undiluted 34.6 34.90 25.81 35.70 GW tertiary 1:2 34.6 34.90 25.81 26.99 GW tertiary 1:4 34.6 34.30 25.81 25.68 GW tertiary 1:8 34.6 34.50 25.81 25.62

102

Sample spiked cDNA sample with Sample Norovirus type Norovirus Enterovirus spiked with Sample Source GII (secondary cDNA GII plasmid Enterovirus ID water plasmid concentrate dilution plasmid control (CT plasmid (CT type control (date) or tertiary (CT mean Mean) mean of (CT Mean) concentrate) of duplicates) duplicates ) Novemb er 28 2015 GW secondary undiluted 31.3 35.30 24.15 29.00 GW secondary 1:2 31.3 34.10 24.15 24.00 GW secondary 1:4 31.3 34.40 24.15 23.90 GW secondary 1:8 31.3 34.30 24.15 23.90 GW tertiary undiluted 31.3 35.30 24.15 29.60 GW tertiary 1:2 31.3 33.80 24.15 24.40 GW tertiary 1:4 31.3 34.50 24.15 23.70 GW tertiary 1:8 31.3 34.40 24.15 24.10 January 2 2016 GW secondary undiluted 31.3 36.60 24.15 32.60 GW secondary 1:2 31.3 34.50 24.15 24.60 GW secondary 1:4 31.3 34.10 24.15 23.60 GW secondary 1:8 31.3 33.70 24.15 23.60 GW tertiary undiluted 31.3 34.60 24.15 32.00 GW tertiary 1:2 31.3 34.70 24.15 24.80 GW tertiary 1:4 31.3 35.50 24.15 24.00 GW tertiary 1:8 31.3 39.60 24.15 23.90 January 13 2016 GW secondary undiluted 31.3 34.90 24.15 29.60 GW secondary 1:2 31.3 33.80 24.15 24.70 GW secondary 1:4 31.3 33.90 24.15 24.10 GW secondary 1:8 31.3 34.60 24.15 24.00 GW tertiary undiluted 31.3 36.00 24.15 32.00 GW tertiary 1:2 31.3 34.70 24.15 26.20 GW tertiary 1:4 31.3 34.60 24.15 24.00 GW tertiary 1:8 31.3 33.80 24.15 23.80 January 30 2016 GW secondary undiluted 31.3 UND 24.15 UND GW secondary 1:2 31.3 34.90 24.15 25.70

103

Sample spiked cDNA sample with Sample Norovirus type Norovirus Enterovirus spiked with Sample Source GII (secondary cDNA GII plasmid Enterovirus ID water plasmid concentrate dilution plasmid control (CT plasmid (CT type control (date) or tertiary (CT mean Mean) mean of (CT Mean) concentrate) of duplicates) duplicates ) GW secondary 1:4 31.3 34.80 24.15 23.70 GW secondary 1:8 31.3 34.10 24.15 24.00 GW tertiary undiluted 31.3 34.70 24.15 31.00 GW tertiary 1:2 31.3 34.40 24.15 25.00 GW tertiary 1:4 31.3 34.60 24.15 23.90 GW tertiary 1:8 31.3 34.20 24.15 23.30 Februar y 4, 2016 GW secondary undiluted 31.3 36.60 24.15 34.40 GW secondary 1:2 31.3 34.30 24.15 25.80 GW secondary 1:4 31.3 34.80 24.15 23.90 GW secondary 1:8 31.3 34.30 24.15 23.80 GW tertiary undiluted 31.3 36.40 24.15 42.30 GW tertiary 1:2 31.3 34.50 24.15 26.60 GW tertiary 1:4 31.3 34.90 24.15 24.20 GW tertiary 1:8 31.3 35.40 24.15 24.20 Februar y 21, 2016 GW secondary undiluted 35.2 35.50 26.10 34.10 GW secondary 1:2 35.2 34.50 26.10 26.70 GW secondary 1:4 35.2 34.90 26.10 26.20 GW secondary 1:8 35.2 34.60 26.10 26.00 GW tertiary undiluted 35.2 35.10 26.10 32.10 GW tertiary 1:2 35.2 34.70 26.10 26.80 GW tertiary 1:4 35.2 35.20 26.10 26.20 GW tertiary 1:8 35.2 34.70 26.10 26.00 March 12, 2016 GW secondary undiluted 35.2 35.20 26.10 32.60 GW secondary 1:2 35.2 35.60 26.10 28.80 GW secondary 1:4 35.2 34.90 26.10 26.20 GW secondary 1:8 35.2 34.90 26.10 26.20

104

Sample spiked cDNA sample with Sample Norovirus type Norovirus Enterovirus spiked with Sample Source GII (secondary cDNA GII plasmid Enterovirus ID water plasmid concentrate dilution plasmid control (CT plasmid (CT type control (date) or tertiary (CT mean Mean) mean of (CT Mean) concentrate) of duplicates) duplicates ) GW tertiary undiluted 35.2 35.70 26.10 32.80 GW tertiary 1:2 35.2 35.50 26.10 27.10 GW tertiary 1:4 35.2 33.80 26.10 26.30 GW tertiary 1:8 35.2 34.60 26.10 26.00 April 3, 2016 GW secondary undiluted 35.2 34.70 26.10 32.20 GW secondary 1:2 35.2 34.60 26.10 27.10 GW secondary 1:4 35.2 35.60 26.10 26.00 GW secondary 1:8 35.2 34.40 26.10 26.00 GW tertiary undiluted 35.2 36.60 26.10 36.80 GW tertiary 1:2 35.2 34.90 26.10 28.10 GW tertiary 1:4 35.2 34.70 26.10 26.20 GW tertiary 1:8 35.2 34.80 26.10 26.00 April 17, 2016 GW secondary undiluted 35.2 34.40 23.90 29.10 GW secondary 1:2 35.2 33.70 23.90 24.90 GW secondary 1:4 35.2 33.80 23.90 24.40 GW secondary 1:8 35.2 34.40 23.90 24.20 GW tertiary undiluted 35.2 34.00 23.90 29.6 GW tertiary 1:2 35.2 33.70 23.90 25.00 GW tertiary 1:4 35.2 34.50 23.90 24.30 GW tertiary 1:8 35.2 33.90 23.90 23.90

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Table 3b Inhibition data for all processed samples from river water (RW) source

Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) January RW 13, 2015 secondary undiluted 33.9 UND 25.412 35.750 RW secondary 1:2 33.9 37.70 25.412 29.359 RW secondary 1:4 33.9 34.90 25.412 27.192 RW secondary 1:8 33.9 34.40 25.412 27.02 RW tertiary undiluted 33.9 UND 25.412 UND RW tertiary 1:2 33.9 34.30 25.412 33.4 RW tertiary 1:4 33.9 33.90 25.412 26.9 RW tertiary 1:8 33.9 33.80 25.412 25.1 RW February 17, 2015 secondary undiluted 33.9 UND 25.3 UND RW secondary 1:2 33.9 UND 25.3 UND RW secondary 1:4 33.9 40.50 25.3 UND RW secondary 1:8 33.9 35.40 25.3 UND RW tertiary undiluted 33.9 UND 25.412 UND RW tertiary 1:2 33.9 UND 25.412 UND

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW tertiary 1:4 33.9 UND 25.412 UND RW tertiary 1:8 33.9 37.00 25.412 34.158 RW March 5, 2015 secondary undiluted 33.9 33.40 25.412 31.647 RW secondary 1:2 33.9 33.90 25.412 26.851 RW secondary 1:4 33.9 34.00 25.412 27.539 RW secondary 1:8 33.9 33.50 25.412 25.974 RW tertiary undiluted 33.9 34.20 25.412 34.322 RW tertiary 1:2 33.9 33.50 25.412 27.067 RW tertiary 1:4 33.9 34.10 25.412 26.529 RW tertiary 1:8 33.9 33.80 25.412 26.027 RW April 14, 2015 secondary undiluted 33.9 35.30 25.412 29.543 RW secondary 1:2 33.9 33.20 25.412 26.15 RW secondary 1:4 33.9 33.50 25.412 25.817 RW secondary 1:8 33.9 33.60 25.412 26.224 RW tertiary undiluted 33.9 UND 34.252 UND

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW tertiary 1:2 33.9 44.40 34.252 UND RW tertiary 1:4 33.9 35.90 34.252 UND RW tertiary 1:8 33.9 33.50 34.252 UND RW May 8 2015 secondary undiluted 33.9 34.30 34.252 UND RW secondary 1:2 33.9 33.20 34.252 43.73 RW secondary 1:4 33.9 33.50 34.252 39.701 RW secondary 1:8 33.9 34.40 34.252 38.113 RW tertiary undiluted 33.9 36.20 34.252 UND RW tertiary 1:2 33.9 33.40 34.252 UND RW tertiary 1:4 33.9 33.40 34.252 43.699 RW tertiary 1:8 33.9 33.50 34.252 40.175 RW June 12 2015 secondary undiluted 33.9 32.90 34.252 UND RW secondary 1:2 33.9 32.50 34.252 43.494 RW secondary 1:4 33.9 32.90 34.252 38.404 RW secondary 1:8 33.9 33.00 34.252 36.465

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW tertiary undiluted 33.9 UND 34.252 UND RW tertiary 1:2 33.9 UND 34.252 UND RW tertiary 1:4 33.9 UND 34.252 UND RW tertiary 1:8 33.9 39.90 34.252 UND RW July 17, 2015 secondary undiluted 33.57 34.11 25.3 35.71 RW secondary 1:2 33.57 32.69 25.3 26.78 RW secondary 1:4 33.57 35.17 25.3 26 RW secondary 1:8 33.57 33.60 25.3 24.458 RW tertiary undiluted 33.57 UND 25.3 UND RW tertiary 1:2 33.57 UND 25.3 UND RW tertiary 1:4 33.57 40.53 25.3 UND RW tertiary 1:8 33.57 34.71 25.3 38.078 RW Sept 15 2015 secondary undiluted 33.57 34.55 25.3 36.106 RW secondary 1:2 33.57 33.18 25.3 30.958 RW secondary 1:4 33.57 33.53 25.3 25.318

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW secondary 1:8 33.57 35.39 25.3 25.315 RW tertiary undiluted 33.57 41.05 25.3 UND RW tertiary 1:2 33.57 36.31 25.3 41.674 RW tertiary 1:4 33.57 33.88 25.3 33.823 RW tertiary 1:8 33.57 33.12 25.3 29.902 RW Sept 30 2015 secondary undiluted 33.57 35.41 25.3 36.523 RW secondary 1:2 33.57 34.22 25.3 25.112 RW secondary 1:4 33.57 33.53 25.3 25.112 RW secondary 1:8 33.57 33.87 25.3 24.758 RW tertiary undiluted 33.57 UND 25.3 UND RW tertiary 1:2 33.57 UND 25.3 UND RW tertiary 1:4 33.57 33.20 25.3 UND RW tertiary 1:8 33.57 38.00 25.3 UND RW Oct 17 2015 secondary undiluted 33.57 34.41 25.3 33.639 RW secondary 1:2 33.57 32.91 25.3 26.782

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW secondary 1:4 33.57 33.54 25.3 25.162 RW secondary 1:8 33.57 34.51 25.3 25.026 RW tertiary undiluted 33.57 UND 25.3 UND RW tertiary 1:2 33.57 UND 25.3 UND RW tertiary 1:4 33.57 36.70 25.3 UND RW tertiary 1:8 33.57 35.41 25.3 32.868 RW Oct 29 2015 secondary undiluted 33.57 38.61 25.3 NA RW secondary 1:2 33.57 34.82 25.3 34.134 RW secondary 1:4 33.57 33.74 25.3 26.734 RW secondary 1:8 33.57 33.73 25.3 26.007 RW tertiary undiluted 33.57 UND 25.3 UND RW tertiary 1:2 33.57 UND 25.3 UND RW tertiary 1:4 33.57 UND 25.3 UND RW tertiary 1:8 33.57 UND 25.3 UND RW Novembe r 14 2015 secondary undiluted 33.50 34.70 25.118 37.477

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW secondary 1:2 33.50 32.80 25.118 28.386 RW secondary 1:4 33.50 32.90 25.118 26.489 RW secondary 1:8 33.50 33.90 25.118 25.863 RW tertiary undiluted 33.50 UND 25.118 UND RW tertiary 1:2 33.50 UND 25.118 UND RW tertiary 1:4 33.50 34.10 25.118 34.819 RW tertiary 1:8 33.50 33.20 25.118 30.701 RW Novembe r 28 2015 secondary undiluted 33.50 34.20 25.118 37.094 RW secondary 1:2 33.50 32.80 25.118 28.787 RW secondary 1:4 33.50 33.40 25.118 26.489 RW secondary 1:8 33.50 33.60 25.118 25.923 RW tertiary undiluted 33.50 UND 25.118 UND RW tertiary 1:2 33.50 37.70 25.118 UND RW tertiary 1:4 33.50 34.10 25.118 37.333 RW tertiary 1:8 33.50 33.40 25.118 31.88

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW January 2 2016 secondary undiluted 33.50 34.50 25.118 38.37 RW secondary 1:2 33.50 33.70 25.118 28.411 RW secondary 1:4 33.50 38.70 25.118 27.435 RW secondary 1:8 33.50 32.80 25.118 28.881 RW tertiary undiluted 33.50 UND 25.118 UND RW tertiary 1:2 33.50 UND 25.118 UND RW tertiary 1:4 33.50 40.40 25.118 UND RW tertiary 1:8 33.50 34.80 25.118 30.616 RW January 13 2016 secondary undiluted 33.50 34.50 25.118 41.613 RW secondary 1:2 33.50 33.30 25.118 30.036 RW secondary 1:4 33.50 38.90 25.118 26.852 RW secondary 1:8 33.50 32.80 25.118 26.301 RW tertiary undiluted 33.50 UND 25.118 UND RW tertiary 1:2 33.50 UND 25.118 UND RW tertiary 1:4 33.50 42.00 25.118 UND

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW tertiary 1:8 33.50 33.50 25.118 39.603 RW January 30 2016 secondary undiluted 33.50 33.80 25.118 37.902 RW secondary 1:2 33.50 32.40 25.118 28.311 RW secondary 1:4 33.50 32.90 25.118 25.531 RW secondary 1:8 33.50 33.40 25.118 26.82 RW tertiary undiluted 33.50 UND 25.118 UND RW tertiary 1:2 33.50 36.90 25.118 UND RW tertiary 1:4 33.50 34.10 25.118 36.705 RW tertiary 1:8 33.50 33.50 25.118 30.616 RW February 4, 2016 secondary undiluted 33.80 34.90 25.3 27.1 RW secondary 1:2 33.80 33.50 25.3 27.1 RW secondary 1:4 33.80 33.70 25.3 24.6 RW secondary 1:8 33.80 33.60 25.3 24.5 RW tertiary undiluted 33.80 UND 25.3 UND RW tertiary 1:2 33.80 UND 25.3 UND

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW tertiary 1:4 33.80 UND 25.3 UND RW tertiary 1:8 33.80 37.10 25.3 UND RW February 21, 2016 secondary undiluted 33.80 34.90 25.3 35.50 RW secondary 1:2 33.80 34.10 25.3 27.8 RW secondary 1:4 33.80 33.90 25.3 24.9 RW secondary 1:8 33.80 33.50 25.3 24.5 RW tertiary undiluted 33.80 UND 25.3 UND RW tertiary 1:2 33.80 42.30 25.3 UND RW tertiary 1:4 33.80 36.00 25.3 40.2 RW tertiary 1:8 33.80 34.00 25.3 30.6 RW March 12, 2016 secondary undiluted 33.80 35.20 25.3 34.4 RW secondary 1:2 33.80 33.50 25.3 27.5 RW secondary 1:4 33.80 34.00 25.3 24.5 RW secondary 1:8 33.80 34.00 25.3 24.3 RW tertiary undiluted 33.80 UND 25.3 UND

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Sample spiked cDNA Sample Norovirus with sample type Enterovirus spiked with Source GII Norovirus Sample (secondary cDNA plasmid Enterovirus water plasmid GII ID (date) concentrate dilution control (CT plasmid (CT type control plasmid or tertiary Mean) mean of (CT Mean) (CT mean concentrate) duplicates) of duplicates) RW tertiary 1:2 33.80 39.40 25.3 UND RW tertiary 1:4 33.80 34.80 25.3 31.6 RW tertiary 1:8 33.80 33.70 25.3 26.6 RW April 3, 2016 secondary undiluted 33.80 37.80 25.3 30.9 RW secondary 1:2 33.80 34.00 25.3 32.1 RW secondary 1:4 33.80 34.00 25.3 29 RW secondary 1:8 33.80 33.80 25.3 25.3 RW tertiary undiluted 33.80 UND 25.3 UND RW tertiary 1:2 33.80 UND 25.3 UND RW tertiary 1:4 33.80 UND 25.3 UND RW tertiary 1:8 33.80 UND 25.3 UND RW April 17, 2016 secondary undiluted 33.80 UND 23.9 32 RW secondary 1:2 33.80 UND 23.9 25.4 RW secondary 1:4 33.80 UND 23.9 24.6 RW secondary 1:8 33.80 UND 23.9 24.4

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Appendix 4: Formulations

MS2 recovery test: Master Mix: 2X PCR Mix (SoFast Syber Green) 10 µL per reaction Water (molecular grade) 0 µL per reaction Primer F 2 µL per reaction Primer R 2 µL per reaction cDNA 6 µL TOTAL VOLUME 20.00 µL

NTC for qPCR: Master Mix (as above) 14 µL, Molecular water or TE buffer 6 µL TOTAL 20 µL qPCR: Master Mix: 2X PCR Mix (applied Biosystems) 2XTAQMAN 12.5 µL per reaction Water (molecular grade) 0.48 µL per reaction Primer F 1 µL per reaction Primer R 1 µL per reaction Probe 0.025 µL per reaction cDNA 5 µL TOTAL VOLUME 20.00 µL

NTC for qPCR: Master Mix (as above) 14 µL, Molecular water or TE buffer 6 µL TOTAL 20 µL

Inhibition test: Plasmid (Enterovirus or Norovirus) 1 µL to each reaction Master Mix 15 µL to each reaction 118

Water (molecular grade) 4 µL to each reaction cDNA Sample 5 µL to each reaction TOTAL 25 µL

NTC for plasmid inhibition: Plasmid 1 µL to each reaction Master Mix 14 µL to each reaction TE buffer 10 µL to each reaction TOTAL 25 µL cDNA PCR product cloning: GOTAQ Hot start master mix was used as directed.

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Appendix 5: Calculations

Calculation used to determine the genomic copies (GC) present in 1L of raw water

GC/L =

Where GC denotes genomic copy, GC is derived from the qPCR software comparing the unknown sample relative the genomic copy in the plasmid standard curve dilution series. 1. Plasmids were extracted and purified 2. 2 µL of plasmid was used to determine the concentration of nucleic material via Nanodrop (in triplicate) ng/ µL (either 1 or 2 µL sample volumes – I used 2 µL) 3. Plasmid extracts with good ratios were selected 4. Using science primer software determined copy number http://scienceprimer.com/copy- number-calculator-for-realtime-pcr 5. Copy number was determined for the plasmid, which was labelled E0 (undiluted) 6. C1V1C2V2, was used to dilute the concentrated E0 to 2.510 7. Serial dilutions, 10-fold, were created from the E10 8. This represents gene copy number per 1 µL as it was derived from Nanodrop ng/ µL concentration 9. qPCR standard curve employed 10 µL of plasmid, therefore the standards must be multiplied by 10 to generate copy number per reaction (the dilution factor approach below is based on copy number per reaction (10 µL). The following values should be entered into the qPCR software: 2500000(E8) 250000 25000 2500 250 25 copy number per reaction 2.5 copy number per reaction (E2)

DF equals the reciprocal of any dilution performed to compensate for inhibition (e.g., 2,4,8 and 16 for 1:2, 1:4,1:8,1:16 and 1:32 dilutions, respectively, or 1 for undiluted samples); And D equals the Volume of Original Water Sample Assayed (see raw data sheet for actual volumes filtered). In general the following was filtered: For GW, 1250 L to 1500 L was filtered For river water, 38 L to ~180 L (one event 360 L) was filtered

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And Y equals the analytical dilutions from the RNA extraction, RT complimentary DNA step and final volume used in qPCR.

Y is derived as follows for tertiary concentrated samples:

* *

= 4 * 7.463 * 2 Y=59.704

Calculation used to determine the approximate Limit of Quantification for Norovirus GII and Enterovirus in Groundwater using tertiary concentrate samples: The limit of quantification is calculated as follows for groundwater samples that detected Norovirus above 3 genomic copies per PCR assay; on average a 1:4 dilution factor for sample volumes of 1250 L was required to overcome inhibition, therefore: 3* [40/10 (4) x 100/13.4 (7.463) x 400/200 (2)] * 4 1250 L = LOQ of 0.6 GC/L for Norovirus GII in groundwater using tertiary concentrates.

The limit of quantification is calculated as follows for groundwater samples that detected Enterovirus above 3 genomic copies per PCR assay; on average a 1:8 dilution factor for sample volumes of 1250 L was required to overcome inhibition, therefore: 3* [40/10 (4) x 100/13.4 (7.463) x 400/200 (2)} * 8 1250 L =LOQ of 1 GC/L for Enterovirus in groundwater using tertiary concentrates.

Calculation used to determine the approximate Limit of Quantification for Norovirus GII and Enterovirus in River water using secondary concentrate samples: Y is derived as follows for secondary concentrated samples:

* *

= 4 * 7.463 * up to 185 Y = up to 5522.62

The range on the limit of quantification is calculated as follows for river water samples that detected Norovirus GII above 3 genomic copies per PCR assay with a median of 1:8 dilution factor (to overcome inhibition) for sample volumes ranging between 38 L and 400 L (average over the study period was 216 L; median was 280 L). 3* [40/10 (4) x 100/13.4 (7.463) x 32000/200 (160)] * 8 280 L =LOQ of 409 GC/L for Norovirus GII in river water

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Therefore the range of LOQs for river water is 286 to 3,016 GC/L for Norovirus GII using secondary concentrates.

The range on the limit of quantification is calculated as follows for river water samples that detected Enterovirus above 3 genomic copies per PCR assay with a median of 1:16 dilution factor for sample volumes ranging between 38 L and 400 3* [40/10 (4) x 100/13.4 (7.463) x 32000/200 (160)] * 16 280 L =LOQ of 818 GC/L for Enterovirus in river water Therefore the range of LOQs for river water is 573 to 6,033 GC/L for Enterovirus using secondary concentrates.

The above factors account for the volume differences between the ten microliters added to the qPCR assay and the volume added to the Vivaspin 20 unit (the Assay Sample Volume).

To reiterate the process, the genomic copies present in the Assay Sample Volume are concentrated to 0.4 mL (400 µL) for tertiary concentrates. The RNA in one-half of that (200

µL) is extracted into a volume of 100 microliters. The RT assay uses 6.7 microliters of the 100

µL in a volume of 40 microliters and then 10 microliters of the RT assay is used for the qPCR assay. Working backwards, the dilution factors are 40/10 (4) x 100/13.4 (7.463) x 400/200 (2)

(ignoring any losses during processing**huge assumption here: ignores sample left in elution tubing and filter; flocculation flask; centrifuge bottles; syringe filter; micropipette tips). Dividing by the volume that D represents the GC/L value for the original sample

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Appendix 6: Jurisdictional scan of water quality standards

Table 6a Jurisdictional Review of selected Drinking Water Standards or Guidelines.

Jurisdiction Legislation/ Matrix: Parameter Value Standard Reference Date Comment Virus policy or Accessed monitoring Potable guideline Drinking (legally Water enforcea  ble or not) WHO WHO (World Drinking E. coli or Not Guideline http://apps. Sept 2016 Concentrations of No Health water thermotolerant present in who.int/iris/ pathogens equivalent guideline Organization) coliform bacteria 100 mL bitstream/1 to a health outcome (2004). sample 0665/44584 target of 10−6 DALY Guidelines /1/9789241 per person per year for Drinking- 548151_en are typically less than water Quality g.pdf 1 organism per 104 – 4th Edition. 105 litres. Sections 7.2 Monitor for indicator Health-based organisms such as E. target coli setting. World Health Organization, Geneva, Switzerland CANADA

123

Jurisdiction Legislation/ Matrix: Parameter Value Standard Reference Date Comment Virus policy or Accessed monitoring Potable guideline Drinking (legally Water enforcea  ble or not) Treated National Guideline for E. coli None Canadian http://www. Sept 2016 Not enforceable unless No potable (Federation Canadian detectabl Drinking hc- provinces have guideline Drinking of provinces) Drinking e per 100 Water sc.gc.ca/ew adopted the guideline for drinking water Water mL Guideline h- into their own water – Quality s – legally semt/alt_for legislative framework treatment enforceab mats/pdf/pu and mandated goal le in some bs/water- compliance provinces eau/sum_g

uide- Enteric protozoa; Treatment res_recom/ Monitoring for Giardia; and goal: sum_guide- Cryptosporidium and Cryptosporidium Minimum res_recom_ Giardia in source 3 log 2014- waters will provide removal 10_eng.pdf valuable information and/or for a risk-based inactivatio assessment of n of cysts treatment and requirements. oocysts Depending on the source water quality, a

greater log removal and/or inactivation may be required Enteric viruses Treatment Routine monitoring for (2011) goal: viruses is not practical; Minimum characterize source 4 log water to determine if reduction greater than a 4 log (removal removal or inactivation and/or is necessary. inactivatio

124

Province of Safe Drinking Regulated E. coli; Not Provincial https://www Sept 2016 No Ontario Water Act drinking detected Act and .ontario.ca/l standard – Total coliform water regulation aws/statute treatment systems - s – legally /02s32 goal Treated enforceab drinking le waters Province of Environment E. coli; Not Provincial http://www. October Treatment standards: No Quebec al Quality Act detected Act and mddep.gou 2016 Groundwater: 99.99% standard – Enterococci regulation v.qc.ca/eau of viruses; treatment bacteria; s – legally /politiq goal

enforceab ue/index- le Surface waters: en.htm 99.99% of viruses, http://www2 99.9% of

125

Jurisdiction Legislation/ Matrix: Parameter Value Standard Reference Date Comment Virus policy or Accessed monitoring Potable guideline Drinking (legally Water enforcea  ble or not) .publication cysts Giardia and Total coliform Not more sduquebec. 99.9% of than 10 oocysts Cryptosporidiu per 100 m mL (within 30 consecuti ve days 90% must not detect total coliform) UNITED The National Public Cryptosporidium; Maximum https://www Sept.2016 No more than 5.0% No STATES OF Primary water Contamin .epa.gov/gr samples total coliform- Maximum Giardia lamblia; AMERICA Drinking systems ant Level ound- positive in a month. Contamina Water Legionella; Goal: water-and- (For water systems nt Regulations drinking- that collect fewer than Level/Stand “zero” Total coliform water/table- 40 routine samples per ard regulated- month, no more than -treatment drinking- one sample can be Treatment goal Viruses water- total coliform-positive goal contaminan per month.) Every 99.9% ts#Microorg sample that has total removal anisms coliform must be analyzed for either fecal coliforms or E. coli if two consecutive TC-positive samples, and one is also positive for E.coli fecal coliforms, system has an acute MCL violation

126

Jurisdiction Legislation/ Matrix: Parameter Value Standard Reference Date Comment Virus policy or Accessed monitoring Potable guideline Drinking (legally Water enforcea  ble or not) State of Group A & B Treated Total coliform; Greater http://www. October Public water systems No Washington Public Water water than one doh.wa.gov 2016 are required to perform standard

Systems sample /Communit routine coliform Rules positive in yandEnviro monitoring. The a month nment/Drin minimum number of kingWater E. coli; Not samples required each detectabl calendar month Faecal coliform e depends on the type of water system, how many people are served, and the source of the water

EUROPEAN EUROPEAN Water E. coli; 0/100 mL http://www. It is neither practical or No UNION UNION intended water.org.u necessary to set Standard Enterococci (DRINKING for human k/sites/defa specific standards for WATER) consumpti ult/files/doc viruses in UK REGULATIO on uments/Ind Regulations or in any NS 2014 supplied ustry%20G revision of the EU by a uidance/vir Drinking Water supplier uses.pdf Directive can be treated or untreated Water, Water E. coli; 0/100 mL In all http://dwi.d Accessed Regulated if supply No England The intended supplies efra.gov.uk/ Sept. 2016 public Standard Enterococci Private water for human stakeholder

Supplies consumpti s/legislation

127

Jurisdiction Legislation/ Matrix: Parameter Value Standard Reference Date Comment Virus policy or Accessed monitoring Potable guideline Drinking (legally Water enforcea  ble or not) Regulation on; /pwsregs20 Clostridium Waters 09.pdf perfringes and influenced spores by surface water Netherlands Groundw Rotavirus and 1.8 x 10 -7 Legislated https://book Sept 2016 Based on acceptable Yes ater polio 3 virus viruses s.google.ca infection risk of one potable per liter /books?id= per 10, 00 persons per finished WPj1CAAA year. Treatment QBAJ&pg= strategies must attain PA57&lpg= the standard PA57&dq= maximum+ allowable+c oncentratio n+and+viru s+and+wat er&source= bl&ots=kc3 CZCkpZo& sig=GohAO 1NUtBS7zj q0rQgX9Yi HiP4&hl=e n&sa=X&ve d=0ahUKE wjm9d-gj- fLAhVFtRQ KHScJDb8 Q6AEIGzA A#v=onepa

128

AUSTRALIA Australian Drinking E. coli; Not No https://www October No guideline value has No Drinking water detectabl regulatory .nhmrc.gov. 2016 been set for Norovirus guideline Enterococci; Water supply e in 100 status. au/_files_n and its inclusion in Thermotolerant Guidelines mL States hmrc/file/pu routine monitoring coliforms; (2011) and blications/n programs is not Version 3.2;– Coliphages territories hmrc_adwg recommended. A Updated implemen _6_februar multiple barrier February t y_2016.pdf approach from 2016 guidelines catchment to tap is recommended to minimise the risk of contamination. Protecting catchments from human and animal wastes is a priority. Operation of barriers should be monitored to ensure effectiveness.

129

Jurisdiction Legislation/ Matrix: Parameter Value Standard Reference Date Comment Virus policy or Accessed monitoring Potable guideline Drinking (legally Water enforcea  ble or not) Drinking E. coli; NEWZEALA New Zealand Less than Standards https://www Accessed Routine monitoring for water ND Ministry of one (or legally .health.govt October pathogenic protozoa is supply Health oocyst/cy enforceab .nz/system/ 2016 not currently Total pathogenic (2000). st) in 100 le files/docum recommended. protozoa Drinking mL of ents/public water sample ations/guid standards for elines- New Zealand drinking- water- quality- manageme viruses No value nt-new- No values have been No set zealand- set due to lack of guideline mar16.pdf reliable evidence

Intended inclusion of a future standard when http://www. the effectiveness of health.govt. viral removal or nz/system/fi inactivation by water les/docume treatment processes is nts/publicati better understood. ons/drinkin g-water- It is considered that if standards- no human effluent is in 2008- the catchment, viruses jun14.pdf will not pose a risk to public health . Available at: www.moh.g ovt.nz/moh.

130

Jurisdiction Legislation/ Matrix: Parameter Value Standard Reference Date Comment Virus policy or Accessed monitoring Potable guideline Drinking (legally Water enforcea  ble or not) nsf/wpg_In dex/Publica tions-Index. New Zealand Ministry of Health (1995). Guidelines for drinking water quality manageme nt for New Zealand.

131

Table 6b Jurisdictional review of selected source water (surface or ground waters) Standards or Guidelines.

Jurisdiction Title Matrix Microbiological Value Standard Reference Date Comment Virus Parameter or Accessed Source Standard or guideline water/ambie Guideline (legally nt (ground enforcea water; ble or surface not) water) WHO Guidelines Surface Guidance Guidance http://www.wh October Code of Good Not for safe waters – o.int/water_sa 2016 Practice for recommended recreational recreational nitation_health Recreational Water water usage /bathing/srwg1 Monitoring has environmen .pdf been developed to ts volume 1 assist with coastal and development of fresh local water quality waters, monitoring 2003 programs.

Recognizes direct human contact and ingestion of water. Protecting Ground None None Guidance http://apps.wh October No specific water Not stated Groundwat waters o.int/iris/bitstre 2016 quality er for am/10665/431 microbiological Health 86/1/92415466 objectives Managing 89_eng.pdf proposed. Difficult the Quality to legally administer of Drinking- –overlapping water jurisdictional Sources boundaries and historical legislation needing reform

132

Jurisdiction Title Matrix Microbiological Value Standard Reference Date Comment Virus Parameter or Accessed Source Standard or guideline water/ambie Guideline (legally nt (ground enforcea water; ble or surface not) water) CANADA Federal Environme Surface None Policy Not stated government ntal waters and statement Protection ground s Act; waters Fisheries Act Recreation Surface E. coli Geometri Guidance http://healthyc September Not al water waters – c mean (5 anadians.gc.c 2016 recommended Guidelines recreational sample a/publications/ usage minimum) healthy-living- ≤ 200 vie- /100mL saine/water- recreational- recreative- eau/index- eng.php Province of Clean Source Provincial October Not stated Ontario Water Act, waters Act and 2016 2006 and intended for regulation rules and drinking s – legally regulations (surface enforceab made water, le under Act ground water) Provincial Surface E. coli 100 per Guidance https://dr6j45jk October Not stated Water waters and 100mL 9xcmk.cloudfr 2016 Quality Ground sample ont.net/docum Guideline waters (geometri ents/3016/moe 133

qualityobjectiv esen.pdf Province of Quebec Groundwater total coliform http://www.md October Quebec Water - for drinking bacteria; — dep.gouv.qc.c 2016 Policy Escherichia coli a/eau/politiq bacteria; — Groundwat ue/index- enterococcus er en.htm bacteria; Catchment http://www2.pu Regulation blicationsduqu under ebec. Environme ntal Quality Act Surface Surface http://www.md October Not stated water: water- delcc.gouv.qc. 2016

sustainable ca/developpe Sustainabili development Faecal coliform Up to 200 ment/indicateu ty policy; per 100 rs/Indicateurs_

Recreation mL DD_Document al water Surface _synthese_ao policy water- ut_2010- recreational en.pdf http://www.md delcc.gouv.qc. ca/eau/criteres _eau/criteres.p df

134

Jurisdiction Title Matrix Microbiological Value Standard Reference Date Comment Virus Parameter or Accessed Source Standard or guideline water/ambie Guideline (legally nt (ground enforcea water; ble or surface not) water) http://www.md delcc.gouv.qc. ca/eau/recreati ve/qualite.htm #conta-micro British Guidance Groundwater Total coliform Screening Guideline http://www2.go Monitoring for Not Columbia Document bacteria, fecal samples v.bc.ca/assets/ microbial hazard to recommended Canada for coliform to gov/environme determine well Determinin bacteria, or determine nt/air-land- assessment (high g Ground Escherichia coli presence water/water/do risk or low risk) Water at (E. coli). cuments/garp_ Risk of assessment_v Containing 2_dec1_2015_ Pathogens final.pdf version 2, November 2015 UNITED STATES OF AMERICA (USEPA) Clean Surface https://www.fs October Federal water Propose Water Act waters ai.ie/uploaded 2016 quality standards coliphage Files/Legislatio found in 40 CFR n/Food_Legisa Part 131.41. tion_Links/Wat

er/SI122_2014 .pdf Individual States can adopt federal

or have authority to https://www.ep meet more 135

water-act EPA is developing

Clean Water Act §304(a) Recreational Water Quality Criteria (RWQC) for coliphage, a viral indicator, to ensure public health protection when recreating in water bodies that may be affected by human fecal contamination

Permits issued to facility that discharge directly into surface waters. Includes municipal and industry USEPA 2012 Surface Geometri Guideline, https://www.ep October Ambient water Not at this time Recreation waters c mean States a.gov/sites/pro 2016 quality criteria

al Water of: have duction/files/20 refers reader to Quality discretion 15- 2012 recreational

Criteria to adopt 10/documents/ water quality

136

L Enterococci https://www.ep a.gov/wqc/nati 30- onal- 35/100mL recommended -water-quality- criteria- human-health- criteria-table New York Water Surface Equal or State https://www.ep October No State, USA Quality waters above standard. a.gov/sites/pro 2016 Testing – If duction/files/20 235 E. Beach exceeded 15- Coli/100m water beach is 10/documents/ L quality closed rec-factsheet- 2012.pdf EUROPEAN UNION DIRECTIV Surface No N/A Guide to http://eur- October Directive is to N/A E waters microbiological member lex.europa.eu/r 2016 contribute to the 2000/60/EC parameters countries esource.html? progressive OF THE to uri=cellar:5c83 reduction of EUROPEA establish 5afb-2ec6- emissions of N respective 4577-bdf8- hazardous PARLIAME regulation 756d3d694eeb substances to NT AND s .0004.02/DOC water. OF THE _1&format=PD

137

of 23 Requires chemical October and biological 2000 characterization establishing only a framework for Community action in the field of water policy COUNCIL Ground No N/A Monitorin http://eur- October Directive prohibits N/A DIRECTIV waters microbiological g of lex.europa.eu/l 2016 or limits the E parameters complianc egal- discharge of certain e with content/EN/TX dangerous of 17 these T/PDF/?uri=C substances into December conditions ELEX:31980L groundwater and 1979 and of the 0068&from=E establishes on the effects of N systematic protection discharge monitoring of the of s on quality of such groundwate groundwa water r against ter is the pollution responsibi caused by lity of the certain competen dangerous t authoritie substances s of the

138

Jurisdiction Title Matrix Microbiological Value Standard Reference Date Comment Virus Parameter or Accessed Source Standard or guideline water/ambie Guideline (legally nt (ground enforcea water; ble or surface not) water) Member (80/68/EEC States ) UK England Groundwat Groundwater None None None http://www.gro October https://www.gov.uk/ None and Wales er undwateruk.or 2016 government/upload

protection: g/Groundwater s/system/uploads/at Principles -issues- tachment_data/file/ and pathogens.asp 297347/LIT_7660_ practice x 9a3742.pdf (GP3),

August 2013 Standards in UK for groundwater follow

zones for source protection based on 50 day attenuation of pathogen entering the zone.

Currently no statutory groundwater quality standards in EU or UK legislation, the chosen environmental standard may be derived using other standards such as the drinking water standard (DWS) or 139

Jurisdiction Title Matrix Microbiological Value Standard Reference Date Comment Virus Parameter or Accessed Source Standard or guideline water/ambie Guideline (legally nt (ground enforcea water; ble or surface not) water) environmental quality standard (EQS). Australia National Surface Intestinal (95th National https://www.nh October Classification None Health and water enterococci percentile guidance mrc.gov.au/_fil 2016 provides incentives Medical s to es_nhmrc/publ for taking action Research intestinal provinces/ ications/attach locally and reducing Council: enterococ regions ments/eh38.pd pollution Guidelines ci/100 mL f for

Managing risks in A= ≤ 40 Recreation B =41– al Water 200 C =201– 500 D => 500 New Zealand Surface E. coli Sample National http://www.mfe October Microbiological None water- 95 guidance .govt.nz/public 2016 assessment Microbiolog Recreational percentile to ations/fresh- category and ical water water - ≤130 E. regional water/microbio sanitary survey quality freshwater coli per agencies logical-water- determine the guidelines 100 quality- grade (A, B, C OR for marine mL(categ guidelines- D) given to the and ory A); marine-and- water. freshwater freshwater- recreational recreatio- areas Sample 14#twoe5 Advise, enterococci Part II: 95 should not be used

140

Jurisdiction Title Matrix Microbiological Value Standard Reference Date Comment Virus Parameter or Accessed Source Standard or guideline water/ambie Guideline (legally nt (ground enforcea water; ble or surface not) water) Section E: percentile because some Publication Microbiolog 131–260 enterococci can date: ical multiply from E. coli per Guidelines June 2003 natural sources, 100mL for such as the decay (B) Freshwater Publication of leaf material. s reference number: Sample 95 ME 474 percentile 261– 550 E. coli per 100 mL(C)

Sample 95 percentile >550 E. coli per 100 mL (D) End of document

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