Enteric virus profiles and adenovirus diversity in municipal wastewater

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The discharge of inadequately treated wastewater into environmental waters such as rivers, lakes and marine waters, which are subsequently used for recreational and/or drinking water sources, can be a human health risk. Water quality is usually monitored through the use of faecal indicator bacteria but as discussed in Chapter 1, they do not necessarily correlate with human enteric viral risk. Therefore, alternative water quality indicators, HAdV and HPyV have been proposed, including for use in faecal source contamination tracking (i.e. FST/MST) (Harwood et al., 2014). Enteric viruses including HAdVs, EVs and/or NoVs, and to a lesser extent HPyVs, have been detected in various water types and multiple locations, most as a result of contamination with sewage or other sources of faecal waste. Examples of studies reporting on virus detection or on disease outbreaks are numerous. These include the detection of enteric viruses in marine water (Griffin et al., 2003; Pina et al., 1998; Puig et al., 1994; Wyn-Jones et al., 2011), ground water (Kukkula et al., 1997; Locas et al., 2007), swimming pool water (D’Angelo et al., 1979; Martone et al., 1980; Sinclair et al., 2009), drinking water (Grabow et al., 2001; Hafliger et al., 2000; Hewitt et al., 2007), storm water (Rajal et al., 2007), surface water (Albinana-Gimenez et al., 2006; Chapron et al., 2000a; Hamza et al., 2014; Jiang et al., 2004; McQuaig et al., 2006; Pusch et al., 2005; van Heerden et al., 2005a; Williamson et al., 2011) including freshwater lakes (Xagoraraki et al., 2007)

Knowledge gaps

The literature review showed that to determine the role of HAdVs and HPyVs as faecal indicators and in predicting the risk of contamination by NoVs, several knowledge gaps would need to be addressed. These were the following: Data on the incidence of HAdVs, HPyVs and NoVs in the New Zealand aquatic environment are limited. Determination of specific HAdV types (both respiratory and enteric) presence in environmental waters was required. Evidence that HAdVs and HPyVs effectively model the occurrence of critical enteric viral pathogens, including NoVs, in diverse aquatic environments was lacking.

Objectives and strategy

To determine the presence and quantity of HAdVs (including specifically HAdV-F, types 40 and 41), HPyVs, NoV GI and NoV GII in surface waters using qPCR. As the detection of HPyVs was not included in Chapter 3, the wastewater samples tested in Chapter 3, along with additional wastewater samples were analysed and results reported here. The WWTPs studied represented those serving different sized communities and geographical locations. To obtain information on the diversity and concentration of enteric and non-enteric/respiratory HAdV types in surface water. Samples where human sewage contamination may have been expected to occur were specifically targeted. The presence of HAdVs and HPyVs were compared to each other and with NoV, and HAdV-F for each sample. Where present, relationships between virus target concentrations were also determined. The data were used to evaluate the suitability of HAdVs and HPyVs as human sewage indicators. RT-qPCR and qPCR assays were used for all test analyses.This chapter is divided into two sections. Section A describes the evaluation of HAdVs and HPyVs as indicators of human sewage contamination in the aquatic environment (published paper). Section B describes the diversity of HAdV types in a range of surface waters.

Section A: Evaluation of HAdV and HPyV as indicators of human sewage contamination in the aquatic environment

Publication details

The following published manuscript forms the basis of this section of the chapter: Hewitt, J. G.E. Greening., M. Leonard and G.D. Lewis. 2013. Evaluation of human adenovirus and human polyomavirus as indicators of human sewage contamination in the aquatic environment. Water Res. 47:6750-6761. This section is as published, except for the following modifications: abstract and acknowledgements were removed with references merged introduction was shortened to avoid repetition abbreviations were modified to be consistent with the rest of the thesis. numbers of tables and figures were modified to fit the formatting of the thesis primers and probes tables used in this study were removed and text added to compensate. references to the methods described elsewhere have been added to the text. changed WWTP F to E, and WWTP H to F, to be consistent with the naming of samples of the WWTP as described in Chapter 3.


Discharge of inadequately treated human wastewater into surface waters used for recreation, drinking water, irrigation and shellfish cultivation may present a public health hazard due to the presence of pathogenic viruses shed mainly from the human gastrointestinal tract (Grabow, 2007; Le Guyader and Atmar, 2007). Other sources of human faecal pollution, including application of biosolids to land, can also contaminate water systems resulting in increased human health risks (Sidhu et al., 2009). Water quality management and human health risk evaluation to assess microbial contamination ideally require effective detection of pathogens. Validation of the use of human enteric viruses as faecal indicators would be useful in water quality assessments and for determining sources of pollution (Colford et al., 2007; Payment et al., 2011; Savichtcheva and Okabe, 2006; Sidhu et al., 2009; Wyn-Jones et al., 2011). Both HAdV and HPyV are extremely common in wastewater throughout the year, with concentrations between 103 and 107 genome copies/L reported in municipal wastewater from different geographical areas (Ahmed et al., 2010; Bofill-Mas et al., 2006; Bofill-Mas et al., 2000; Bofill-Mas et al., 2010b; Haramoto et al., 2010; McQuaig et al., 2009). As shown in Chapter 3, NoVs excreted in large amounts in the faeces of infected individuals can be present in high concentrations (>103 genome copies/L) in municipal wastewater. As NoVs are not persistently excreted by humans, and with outbreaks often showing seasonal tendencies, their presence in wastewater may be more sporadic than HAdVs and HPyVs. In wastewater samples taken from treatment plants serving small populations, the variation in virus concentrations may be more apparent (Hewitt et al., 2011). In addition, during periods of high excretion by the population, prevalence or concentration of enteric pathogens such as NoV or rotaviruses may be greater than potential faecal indicators such as HAdVs and HPyVs (Miagostovich et al., 2008). NoV can be present at high concentrations in wastewater from populations with no or few reported NoV outbreaks (Hewitt et al., 2011) and so since in New Zealand NoV outbreaks do not demonstrate any consistent seasonal tendencies (Greening et al., 2012), it is expected that NoVs could be prevalent in wastewater samples and hence receiving waters throughout the year. Although primarily transmitted person to person, NoVs are responsible for acute gastroenteritis outbreaks associated with faecally contaminated water, shellfish and other foods due mainly to their environmental stability (Lopman et al., 2012) and low infectious dose (La Rosa et al., 2012; Teunis et al., 2008). Faecal indicator bacteria are often used in water quality management but can fail to predict the presence of pathogens such as NoVs that are more environmentally resistant than the indicator.

Viruses and samples

HAdV-2, HAdV type 41, and NoV GI and GII positive samples as previously described (Hewitt et al., 2011) were used as (RT)-qPCR positive controls (Appendix A). The HPyV qPCR positive control was derived from a HPyV JC strain recovered from an influent wastewater sample and identified by DNA sequencing of the PCR product (McQuaig et al., 2009). Primary screened influent wastewater, treated effluent wastewater, river water, urban stream water and estuarine water were collected from sites around New Zealand between December 2003 and June 2013. Samples were initially collected to provide information on virus input into and removal from WWTP, for water quality monitoring programmes and risk assessments. It was therefore likely that human enteric viruses would be present in many of the samples. Ten pairs of wastewater influent and effluent samples were taken from seven WWTP, with an additional 15 effluent samples taken from a further four WWTP. Most effluent samples were collected before any mechanical or chemical disinfection processes. Despite the paired samples being taken on the same day, they were temporally separate due to the time required for the wastewater to pass through the plant. WWTP processes and the population size served by each plant varied (Tables 5.2 and 5.3). Several samples (two influents and seven effluents) were as described in Chapter 3 and are identified in Tables 5.1 and 5.2.Water samples were collected from two New Zealand rivers. One site on each river was used for sampling every two weeks over a two-year period. River site 1 was in the North Island of New Zealand and represented a river with urban impacts. River site 2 was in the South Island of New Zealand and represented a mainly agriculturally impacted river. The river water samples (n=35) used for this study were selected from a total of 109 samples collected from these two sites between 2007-2009, and previously analysed for several enteric viruses including HAdVs, NoV GI and NoV GII (Williamson et al., 2011). Of these 109 samples (of which 71 (65%), 36 (33%) and 66 (61%) were HAdV, NoV GI and NoV GII positive respectively), 30 HAdV positive samples and five HAdV negative samples were chosen for HAdV-F and HPyV analysis. Water samples (n=21) were collected from seven urban streams impacted by combined sewage overflows and were located in a city in the North Island. Water samples (n=15) were collected from several sites within one harbour estuarine area in the North Island. Following three separate high rainfall events that resulted in primary treated wastewater overflows from a nearby WWTP that serves a population of >50,000 into the catchment area, samples were taken from each of the sampling sites within one day of each event.

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Sample processing and virus recovery

Viruses were recovered from influent and effluent wastewater samples (1 L) collected in 2003 and 2004, as previously described where the method had a recovery efficiency of at least 10% (Hewitt et al., 2011). For all other samples (2-20 L) collected from 2007 to 2010, Hemoflow HF80S dialysis ultrafilters (Fresenius Medical Care) were used to concentrate the sample volume to approximately 0.5 L (Appendix E.3). Viruses were eluted from the solid fraction with 3% (wt/vol) beef extract-0.05 M glycine solution (pH 9.0) and with the viruses recovered from the liquid fraction, concentrated further using PEG precipitation (Hewitt et al., 2007). From 2011 to 2013, FX80 dialysis ultrafilters (Fresenius Medical Care) were used instead due to the unavailability of HF80S filters. Due to the diverse sample types and large variability among the sample type matrices, river waters from two sources were used as representative samples to determine HAdV and NoV recoveries using the ultrafilters. HAdV type 2, NoV GI and NoV GII were seeded into 10 L samples and virus recovery (%) determined. HPyV recovery was not determined due to stock unavailability. Using the Hemoflow HF80S filters, mean recoveries calculated from undiluted samples ranged from 4% for HAdV type 2 to 34% for NoV GII (as previously described in Williamson et al., 2011). In trials comparing NoV and HAdV recoveries using HF80S and FX80 dialysis ultrafilters, comparable quantities (genome copies) of viruses were recovered from representative effluent wastewater samples (data not shown).

Nucleic acid extraction, RT-qPCR and qPCR

Following virus recovery, viral nucleic acid was extracted from 200 µL of the sample concentrates using the High Pure Viral Nucleic Acid Kit (Roche Molecular Biochemicals Ltd.) as per manufacturer’s instructions. All samples were analysed for HAdV, HAdV-F, HPyV, NoV GI and NoV GII. Two generic HAdV qPCR assays were used: HAdV assay #1 (modified from Heim et al., 2003) for the river water samples and HAdV assay #2 (Hernroth et al., 2002) for all other matrices. Two generic NoV GII qPCR assays were used: NoV assay #1 (modified from Wolf et al., 2010) for the river water samples and NoV assay #2 (Kageyama et al., 2003) for all other matrices. HAdV, HAdV-F, HPyV, NoV GI and NoV GII quantification (genome copies per reaction) was determined by comparing the PCR cycle threshold value against a linear standard curve generated from log10 dilution series of 107 to 10 genome copies per reaction of the appropriate DNA plasmid prepared from PCR products (as previously described in Wolf et al., 2010). PCR titres per litre of sample were calculated using the concentration factor and amount of sample analysed in each PCR. The data was then log10 transformed to express the concentration as log10 genome copies/L. In each (RT)-qPCR assay, virus positive and negative (water) extraction controls, specific DNA plasmid controls, and DNase/RNase free water as a non-template control were included.

1.1. Study aims
1.2. Enteric viruses in the aquatic environment
1.3. Faecal indicators, indices and surrogates
1.4. Role of viruses and bacteriophages as indicators
1.5. Human adenoviruses
1.6. Human polyomaviruses
1.7. Key knowledge gaps
1.8. Study objectives
1.9. Overall study strategy
2.1. Objectives and strategy
2.2. Methods literature review
2.3. Key considerations
2.4. Methodological areas for development
2.5. Method development strategy
2.6. PCR development
2.7. HAdV cell culture development using guanidine hydrochloride
2.8. Development of a virus recovery method from water
3.1. Background
3.2. Knowledge gaps
3.3. Objectives and strategy
3.4. Section A: Influence of wastewater treatment process and the population size on human virus profiles in wastewater
3.5. Section B: Diversity of HAdV in influent and effluent wastewater
4.1. Introduction
4.2. Knowledge gaps
4.3. Objectives and strategy
4.4. Methods
4.5. Results
4.6. Discussion
5.1. Knowledge gaps
5.2. Objectives and strategy
5.3. Section A: Evaluation of HAdV and HPyV as indicators of human sewage contamination in the aquatic environment
5.4. Section B: Diversity of HAdV in surface waters
6.1. Introduction
6.2. Knowledge gaps
6.3. Section A: HAdV and HPyV presence in shellfishCHAPTER
6.4. Section B: Method evaluation for HAdV recovery
6.5. Section C: Diversity of HAdV in shellfish
APPENDIX B. Cell lines
APPENDIX C. Reagents (non-PCR)
APPENDIX D. Primers and probes
APPENDIX E. Virus recovery methods from waters
APPENDIX F. Viral nucleic acid extraction and PCR methods
APPENDIX G. Cell culture methods
APPENDIX H. PCR product purification and DNA sequencing
APPENDIX I. DNA plasmid preparation and quantification
APPENDIX J. Determination of virus recovery
APPENDIX K. Statistics


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