Genetic diversity and quantification of human mastadenoviruses in wastewater from Sydney and Melbourne, Australia
Graphical abstract
Introduction
Enteric viruses are of major concern due to their low infectious dose, high viral shedding in patients, and stability in external environments (Rodriguez-Lazaro et al., 2012). Viral water contamination can occur when human and animal waste enter waterways without proper, or insufficient, treatment for pathogen removal. Point source (combined sewer overflow and sanitary sewer overflow) and non-point source (i.e. agricultural runoff, atmospheric pollution etc.) contamination causes environmental human viral pollution from treated wastewater discharges. This can pose a significant public health risk to swimmers and recreational users, and has the potential to infect many people when drinking water or food sources are contaminated (Kukkula et al., 1997; Mac Kenzie et al., 1994).
Acute gastroenteritis (AGE) is the second leading cause of death in children under the age of five, resulting in approximately 1.3 million annual deaths (Wang et al., 2016). Symptoms of AGE include diarrhoea, vomiting, abdominal pain, myalgia, nausea, chills and fever. Despite improved global food safety and hygiene practises, AGE continues to have a significant health impact on people of all ages, in both developed and developing countries. Even though viral gastroenteritis is generally self-limiting, severe dehydration can lead to death in more vulnerable individuals (Echavarría, 2009; Payne et al., 2013; Siebenga et al., 2009). Outbreaks of viral gastroenteritis are difficult to control and commonly occur in hospital wards, cruise ships, childcare centres and aged care facilities (Maunula et al., 2017; Jones et al., 2018).
Viruses commonly associated with waterborne disease outbreaks include human mastadenovirus (HAdV), norovirus, rotavirus (RoV), hepatitis A virus (HAV), hepatitis E virus (HEV) and enterovirus (Kukkula et al., 1997). These enteric viruses are transmitted through the faecal-oral route and can affect the gastrointestinal tract or the liver. Noroviruses are the leading cause of viral gastroenteritis, accounting for approximately 50% of global gastroenteritis cases (Payne et al., 2013), and enteric HAdV causes up to 15% of all AGE (Chatterjee et al., 2004; Froggatt et al., 2004), whilst HAV and HEV together are responsible for 4.1% of viral hepatitis mortality (Jacobsen and Wiersma, 2010; Rein et al., 2012).
HAdVs are non-enveloped, double-stranded icosahedral DNA viruses, approximately 90–100 nm in diameter, with fibre projections on each of the 12 vertices (Davison et al., 2003). The genome is approximately 30–35 kilobases in length and encodes more than 30 structural and non-structural proteins (Friefeld et al., 1984). HAdV belongs to the genus Mastadenovirus, one of five genera within the Adenoviridae family. Within Mastadenovirus, seven known groups have been identified (A-G), which can be further classified into 54 serotypes (Ghebremedhin, 2014).
In addition to gastroenteritis, HAdV can cause a wide range of clinical diseases, including respiratory illness, conjunctivitis, hepatitis and pneumonia (Ghebremedhin, 2014). This is because many HAdV groups, in particular group D, have a wide tissue tropism. Of all seven groups, groups A, F and G have selective tropism for the gastrointestinal (GI) tract and are rarely found to infect other tissues (Chang et al., 2008; Murphy et al., 2012; Walls et al., 2003). Whilst groups B, C and E are more commonly associated with respiratory illnesses (Chang et al., 2008), they can also cause gastroenteritis (Adrian and Wigand, 1989; Brown, 1990). Group D viruses mainly cause conjunctivitis (Chang et al., 2008; Maranhão et al., 2009; Sambursky et al., 2007).
The quantification of pathogenic viruses in wastewater is important to understand the viral burden and possible risks involved in the case of viral contamination of water sources, as this can pose a significant public health problem and affect hundreds of people. Furthermore, knowledge of enteric virus levels can help determine the necessary treatments required for proper removal of pathogenic viruses in the production of recycled water.
Despite the number of studies performed to measure the DNA levels of HAdV in untreated wastewater, several issues have been identified. Firstly, the concentration of total virus measured by previous studies varies considerably, ranging from 6.3 × 104 (Ogorzaly et al., 2015) to 6.5 × 107 genome copies/L (Bofill-Mas et al., 2013). Secondly, the measurement unit used is not standardised between studies; units used include non-standardised polymerase chain reaction (PCR) detectable units (PDU)/L (Lodder and de Roda Husman, 2005; van den Berg et al., 2005) and reverse-transcription (RT)-PCR or PCR units/L (Katayama et al., 2008), rather than genome copies/L. This prevents any direct comparison between these studies. Thirdly, the majority of published studies have not included a process control to ensure all steps performed in the quantification assay are validated, including concentration and nucleic acid extraction processes (Ogorzaly et al., 2015; Fong and Lipp, 2005; Prado et al., 2011). Therefore, there is a need for better methods and controls for quantification of enteric viruses within wastewater systems, which this study has addressed.
Despite the large number of molecular epidemiological studies on human enteric viruses, the majority have been conducted with clinical samples from patients presenting with gastroenteritis or hepatitis. HAdV-associated gastroenteritis is generally asymptomatic or self-limiting, and usually does not require hospitalisation or special medical attention (Griffin et al., 2003). Consequently, HAdV-associated clinical samples are inherently biased towards symptomatic/more severe cases and so are not representative of all HAdVs in the population.
During the symptomatic phase of infection, high levels of virions can be found in stools of infected individuals and viral particles continue to be shed for months after symptoms have subsided (Radke and Cook, 2018). Additionally, enteric viruses are very stable in external environments and resistant to degradation (Thurston-Enriquez et al., 2003). The combination of these two factors leads to high levels of enteric viruses in wastewater, and as such, represents a useful source to comprehensively study the molecular epidemiology of virus populations within the community.
To date, only a limited number of studies have examined the correlation between genotypic diversity of HAdV between wastewater and clinical samples (Ogorzaly et al., 2015; Prevost et al., 2015). Of those studies, the majority do not quantify the total number of viruses, and older Sanger sequencing technologies have been used to measure viral diversity (Lodder and de Roda Husman, 2005; Prado et al., 2011; Pusch et al., 2005). These traditional cloning and sequencing methods are biased towards the more prevalent strains in a given sample, and do not give an accurate overall picture of the genetic diversity in wastewater systems (Iaconelli et al., 2017). With the advancements in next generation sequencing (NGS) technology, HAdV serotype diversity can now be determined and monitored in complex wastewater samples for a more comprehensive understanding of viral molecular epidemiology at the population scale.
The aim of this study was to quantify concentrations and diversity of HAdV in three wastewater treatment plants in Sydney and Melbourne, Australia. With the use of molecular methods, the HAdV DNA was quantified to better understand viral levels within the wastewater systems. Furthermore, using NGS technologies, the diversity of HAdV was determined in wastewater representing millions of people, i.e. at a population level. The diversity of HAdV identified in wastewater was then compared to those found in clinical HAdV data from New South Wales (NSW) to determine if the groups and serotypes identified between the two sample types were similar.
Section snippets
Wastewater sample collection
A total of 20 monthly influent samples (24-h composites) were collected by autosampler from wastewater treatment plants (WWTP) in Sydney (Bondi and Malabar) between January 2016 and December 2017, with a population capacity of 296,350 and 1,667,460, respectively. For Melbourne (Western Treatment Plant), composite (flow-weighted) influent samples were collected monthly by autosampler from January to December in 2016, and May to December in 2017 (population capacity 2,196,380). All samples were
Quantitative analysis of HAdV in wastewater samples
A total of 68 monthly wastewater samples were collected from three WWTPs over the study period. Using qPCR, HAdV DNA levels were measured and expressed as genome copies per litre. MS2 bacteriophage was used as an extraction control for all downstream processes, all samples used in this study obtained a cycle threshold (ct) value of 16.3 ± 1.6 using this control.
In the Bondi WWTP, the highest HAdV DNA levels were observed in the summer season between January and March of both 2016 (average of
Discussion
The combination of low infectious dose, high viral shedding and stability in the external environment enables the persistence of enteric viruses within a population. The use of wastewater for enteric virus surveillance enables a non-biased identification of viral genetic diversity within the community.
The concentration of infectious HAdV can be determined using cell-culture methods, however, the use of different host cell lines in these assays is biased due to the tropism of different serotypes
Conclusion
The quantification of enteric viruses within the wastewater system can enhance our knowledge and identify potential risks associated with viral contamination of receiving environments in the case of inadequate treatment or unplanned sewage releases. The HAdV DNA level was determined in wastewater samples collected from Sydney and Melbourne, Australia. An average of 1.8 × 107 genome copies/L were identified for HAdV. Furthermore, as the majority of HAdV infections are asymptomatic, a more
Acknowledgments
We thank our colleagues Alper Yasar and Jason Koval from the Ramaciotti Centre for Genomics (UNSW) for advice and technical support with MiSeq sequencing. We would like to thank Professor William Rawlison and Juan Merif (Prince of Wales Hospital) for provision of clinical samples. We would also like to thank Melbourne Water Corporation and Sydney Water Corporation for provision of wastewater samples.
Funding
This Australian research was supported by funding from Melbourne Water Corporation and the National Health and Medical Research Council (project no. APP1083139 and APP1123135). Jennifer Lun was supported by an Australian Postgraduate Award and a Water Research Australia Scholarship.
Conflicts of interests
Jennifer Lun's Water Research Australia scholarship was funded by Melbourne Water Corporation, and this work was partially funded by Melbourne Water Corporation.
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