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Waterborne human pathogenic virusesof public health concernAtheesha Ganesh a & Johnson Lin aa Discipline of Microbiology, School of Life Sciences, University ofKwaZulu-Natal (Westville), Durban, South AfricaVersion of record first published: 22 Feb 2013.

To cite this article: Atheesha Ganesh & Johnson Lin (2013): Waterborne human pathogenicviruses of public health concern, International Journal of Environmental Health Research,DOI:10.1080/09603123.2013.769205

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Waterborne human pathogenic viruses of public health concern

Atheesha Ganesh and Johnson Lin*

Discipline of Microbiology, School of Life Sciences, University of KwaZulu-Natal (Westville),Durban, South Africa

(Received 20 March 2012; final version received 16 December 2012)

In recent years, the impending impact of waterborne pathogens on human health hasbecome a growing concern. Drinking water and recreational exposure to pollutedwater have shown to be linked to viral infections, since viruses are shed in extremelyhigh numbers in the faeces and vomit of infected individuals and are routinely intro-duced into the water environment. All of the identified pathogenic viruses that posea significant public health threat in the water environment are transmitted via the fae-cal–oral route. This group, are collectively known as enteric viruses, and their possi-ble health effects include gastroenteritis, paralysis, meningitis, hepatitis, respiratoryillness and diarrhoea. This review addresses both past and recent investigations intoviral contamination of surface waters, with emphasis on six types of potentialwaterborne human pathogenic viruses. In addition, the viral associated illnesses areoutlined with reference to their pathogenesis and routes of transmission.

Keywords: waterborne; pathogen; virus; gastroenteritis; pathogenesis

Introduction

Viruses are omnipresent and extraordinarily abundant in the microbial ecosystems ofwater, soil and sediment (Wommack et al. 1995). In nearly every reported case foraquatic and porous media environments, (soils and sediments) viral abundance exceedsthat of co-occurring host populations by 10-100-fold (Wommack et al. 2009). If currentestimates based on meta-genomic DNA sequence data are correct, then viruses representthe largest reservoir of unknown genetic diversity on Earth (Wommack et al. 1995;Williamson et al. 2008).

Surface water can be contaminated with enteric viruses by a variety of sources.Enteric viruses are shed in extremely high (1011/g faeces) numbers in the faeces andvomit of infected individuals (Fong and Lipp 2005) and are routinely introduced intothe environment through the discharge of treated and untreated wastes (de Roda Hus-man and Bartram 2007). In consequence, viral pathogens in vomit and faeces ofinfected individuals contaminate the marine environment, fresh water and groundwater.These viruses contaminate drinking water sources, recreational waters and irrigationwaters, thereby enabling viral transmission from person-to-person and surface-to-personto occur (Griffin et al. 2003). Since viruses cannot multiply outside a living host andare exceptionally resistant to unfavourable conditions (Hollinger and Emerson 2007),virus levels tend to decrease gradually after discharge into the aquatic environment and

*Corresponding author. Email: linj@ukzn.ac.za

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may be present in water for a long period (Okoh et al. 2010). Mankind is exposed tothese enteric viruses through various means: shellfish grown in polluted waters, contam-inated drinking water and food crops grown in land irrigated with sewage contaminatedwater and/or fertilised with sewage. Foods susceptible to be contaminated at the pre-harvest stage have also been implicated in outbreaks of viral diseases (Bosch et al.2008). Possible health effects associated with the presence of such viruses in waterinclude paralysis, meningitis, hepatitis, respiratory illness and diarrhoea (Villena et al.2003; Hewitt et al. 2007).

Current treatment practices are unable to provide virus-free wastewater effluents(Dongdem et al. 2009). Wastewater treatment processes pertaining to the activatedsludge process, oxidation ponds, activated carbon treatment, filtration, and lime coagula-tion and chlorination merely eradicate between 50 and 90% of viruses present in waste-water (Okoh et al. 2010), thus allowing for a significant viral load to be released ineffluent discharge. Due to their steadiness and perseverance, enteric viruses conse-quently become pollutants in environmental waters resulting in human exposure throughpollution of drinking water sources and recreational waters, as well as foods. Drinkingwater, if ineffectively treated, can contain enteric viruses such as human adenoviruses(HAdVs) and noroviruses (NoVs) derived from source water and pose a health risk topeople on consumption (Fong and Lipp 2005). The inherent resistance of enteric virusesto water disinfection processes means that they may likely be present in drinking waterexposing consumers to the likelihood of infection (Okoh et al. 2010). Research carriedout in Germany showed that even though microbiological parameters such asEscherichia coli, enterococci and coliphages indicated acceptable microbiological waterquality, the virological data suggested that surface waters might still be sources forenteric viral infections (Pusch et al. 2005). Viruses are accountable for 14% of gastroen-teritis (GE) occurrences and 38% of illnesses associated with drinking water in theUnited States (US) from 1999 to 2002 (Li et al. 2010) and are on the “contaminant can-didate list” of the US Environmental Protection Agency (USEPA) for regulatory consid-eration for drinking water programme (Federal Register 2005). The outbreak ofwaterborne viral diseases has been reported extensively worldwide (Ye et al. 2012).Approximately 655,965 human cases of infectious diarrhoea (excluding cholera, dysen-tery, typhoid and paratyphoid) were reported in China during 2009, with 33,087 casesbeing diagnosed definitely in aetiology, of which 92.79% were caused by virus infection(Ye et al. 2012). Rotavirus (RV) was the main infectious agent accounting for 92.58%of the diarrhoeal cases, tailed by adenovirus (AdV) accounting for 3.29% (Ma et al.2010). Simultaneously, there were 488,955 cases of hand, foot and mouth disease inChina during 2008 and 1155,525 cases in 2009 which were primarily caused by entero-virus (EV) 71 and coxackievirus A16 (Chang et al. 2011). During this period, 0.2–1.2%of the diseased cases expressed severe symptoms, with 2.6–10.8% being fatal (Changet al. 2011).

Human enteric viruses, which primarily infect and replicate in the gastrointestinaltract, have been associated with waterborne transmission (Carter 2005; Grabow 2007)and therefore have the potential to pollute surface (Pintó and Saiz 2007), ground (Gerba2007) and drinking water (Carter 2005). Enteric viruses can cause a meaningful risk ofinfection in vulnerable individuals even at low levels of viral pollution (Fong and Lipp2005; Teunis et al. 2008). The high prevalence of enteric viruses in surface water high-lights the importance of assessing the water sources used for domestic purposes forviral contamination (Kiulial et al. 2010). In South Africa, the government loses aboutR3.4 billion annually, due to approximately three million diarrhoeal cases and 50,000

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mortalities (Momba et al. 2010). Diarrhoea is also considered as a signature hallmark ofHIV/AIDS because over 80% of patients in developing countries suffer chronic diar-rhoea (Momba et al. 2010). A recent survey across seven of the nine provinces ofSouth Africa revealed the failure of the majority of these water treatment plants to pro-duce drinking water at the points of treatment and in distribution systems (Mombaet al. 2009). The surveillance of surface water samples for enteric viruses becomes animportant indicator of the level of human faecal pollution regardless of the socioeco-nomic status of a country. This information can be used to further assess the publichealth risks associated with exposure to these water sources (van Heerden, Ehlers,Vivier et al. 2005; Venter et al. 2007; Espinosa et al. 2008). This review presents anoverview of the role of enteric viruses as waterborne pathogens of humans, and therisks presented from waterborne exposure. Specific discussions are focused on six viralgroups, which are among the most well studied in terms of waterborne contamination.

Viral studies in water environments

Research in aquatic viral ecology has mainly focused on marine microbial ecology. Ittook only a few years before other environments such as lakes, rivers, sediments, aswell as soils, to be studied from a virological perspective (Suttle 2005, 2007). Virusesare essential members of aquatic ecosystems and appear to be not only the most abun-dant but also the most diverse biological entities (Angly et al. 2006).

In freshwater ecosystems, estimates of the abundance of viruses (or virus-like parti-cles) have only recently begun to be documented (Wilhelm and Matteson 2008). In part,this may be attributable to the development of more feasible approaches to enumeratingtotal virus abundance by epifluorescence microscopy (Wen et al. 2004). Some studiessuggest that enteric virus abundance in freshwaters tend to be higher relative to marineenvironments (De Bruyn et al. 2004; Filippini and Middelboe 2007). In marine environ-ments, the roles of planktonic viruses as regulators of carbon and nutrient cycling aswell as microbial community structure have been a focus of numerous studies, yet theroles of freshwater virioplankton remain less studied (Suttle 2007). Fluctuations in nutri-ent concentrations, temperature and community structure tend to be more significantand predictable factors (primarily due to strong seasonal cycles) in the detection ofviruses in aquatic ecosystems (Lymer et al. 2008; Wilhelm and Matteson 2008;Sawstrom et al. 2009; Pradeep and Sime-Ngando 2010). Understanding the regulationand dynamics at various spatial and temporal scales is crucial to realise the viralecology in the freshwater environment and their impact factors such as climate change.The predominant factors affecting virus survival in the water environment are tempera-ture, virus association with solids, exposure to ultraviolet (UV) and the presence ofmicrobial flora (Bosch et al. 2008). The effect of temperature on viral perseverance inwater may be due to several mechanisms including viral protein denaturation, RNAdamage and influence on microbial or enzymatic activity (Bosch et al. 2008).

In a water setting, the migration of enteric viral pathogens may take several proba-ble routes: ill human! human faeces! sewage!wastewater treatment plant! riverwater receiving sewage!water intake!water treatment process! tap water (Kovaćet al. 2009). Several studies have pointed out the role of groundwater as a source ofviral outbreaks in countries of different economic level (Fong et al. 2005). Entericviruses are the most probable human pathogens to contaminate groundwater, due totheir extremely small size, which allows them to penetrate soils from contaminationsources such as broken sewage pipes and septic tanks, eventually reaching aquifers

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(Okoh et al. 2010). EVs, NoVs, RVs and hepatitis A viruses (HAVs) have been detectedin many groundwater supplies, including private household wells, municipal wells andunconfined aquifers (Borchardt et al. 2003). In these above studies, surface waters orseptic tanks were identified as the most probable sources of contamination. Groundwa-ter has been associated as a common transmission route for waterborne transferable dis-ease in the US with about 80% of viral waterborne outbreaks being attributed todrinking contaminated well water (Borchardt et al. 2003). The enteric viruses regularlyassociated with these outbreaks were NoVs and HAV. Potential adsorbents of viruses innatural waters include sand, pure clays (e.g. montmorillonite, illite, kaolinite and ben-tonite), bacterial cells, naturally occurring suspended colloids, and estuarine silts andsediments (Okoh et al. 2010). Removal rates depend to a great extent on the pH, sub-strate saturation, redox potential and dissolved oxygen of the system.

In addition to the whole community approach, studies in specific virus-host interac-tions are also important in further understanding the diversity, dynamics and regulationof viruses and host populations present in freshwater environments. There are manyimportant gaps such as ecological consequences of viral genetic diversity and the influ-ence of viral activity on host diversity. Additionally, biogeochemical cycles still need tobe explored to comprehend the importance in genetic and functional diversity in viralcommunities (Middelboe et al. 2008). Development of tools for analysing viruses inaquatic ecosystems is thus essential for obtaining accurate measurements of their activ-ity and for predicting the consequences of these activities (Miki et al. 2008).

Waterborne human pathogenic viruses of public health concern and theirassociated illnesses

Viruses represent the planet’s largest pool of genetic diversity and human pathogenicity(Rosario et al. 2009). All of the identified human pathogenic viruses that pose a signifi-cant public health risk in the water environment are transmitted via the faecal–oral route(Griffin et al. 2003). The significance of enteric viruses as causative agents of crucialhuman diseases cannot be overrated. These viruses belong primarily to the familiesAdenoviridae (AdV strains 40 and 41), Caliciviridae (Norwalk virus, astroviruses(AstVs), caliciviruses and small round structured viruses), Picornaviridae (poliovirus(PV), coxsackieviruses, echoviruses, EVs and HAV) and Reoviridae (reoviruses andRV). These enteric viruses are associated with a variety of diseases in humans, such asocular and respiratory infections to GE, hepatitis, myocarditis and aseptic meningitis(Griffin et al. 2003). The primary site of viral infection and replication is the intestinaltract.

Diarrhoeal diseases affect millions of people around the world and have the greatestimpact on children, especially those in developing countries (Moe and Rheingans2006). Diarrhoea is one of the leading causes of death in developing countries, respon-sible for 25–30% of deaths among children younger than five years of age (Martineset al. 1991). In these countries, the incidence of diarrhoeal cases varies between 2.5 and3.9 episodes per child per year. Almost every child contracts a diarrhoeal disease duringthe first five years of their life, on an average several times per year (Bern and Glass1994). In South Africa, diarrhoeal disease is a leading cause of childhood mortality,being responsible for 10% of all causes of death in 2000. A Cochrane review on thesubject concluded that interventions to improve water quality are generally effective inpreventing diarrhoea (Clasen et al. 2007). Diarrhoea can be caused by a number of dif-ferent agents, including viruses, bacteria, parasites and toxins. However, during the past

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few decades, viruses have been firmly established as aetiological agents of acute GE(Bern and Glass 1994). GE is a communicable disease characterised by fever,abdominal cramps, nausea, vomiting, diarrhoea and headache. RV are the leading causeof severe diarrhoeal disease and dehydration in infants and children under the age of 5worldwide (Parashar et al. 2003; Van Damme et al. 2007). Hepatitis, another contributorto waterborne disease, can be a seriously debilitating disease progressing from non-specific illnesses with fever, headache, nausea and malaise to vomiting, diarrhoea,abdominal pain and jaundice. HAV represents globally approximately 50% of the totalhepatitis cases and although the disease is self-limiting and rarely causing death, it mayincapacitate patients for several months (Pinto and Saiz 2007).

Human enteric viruses are detected frequently in various water samples constitutinga primary source of GE outbreaks (Rutjes et al. 2005). Since the presence of a few viralparticles is sufficient to cause disease, detection of low concentration of these viruses inenvironmental samples or in food matrices is usually complex. Therefore, the methodsfor detection of viruses on food and water samples must have a high level of sensitivityand specificity (Bosch et al. 2011). A wide range of detection methods are available fordetection of environmental viruses. Growth in tissue culture and plaque assay for enu-meration is the gold standard for infectious virus quantification (Mattison and Bidawid2009). However, the conditions under which many enteric pathogens can be culturedare not known (Mattison and Bidawid 2009), and other viruses grow slowly or do notproduce a cytopathic effect (Bosch et al. 2011). Other methods such as electron micros-copy (EM) and immunoelectron microscopy to directly visualise viral particles providean alternative approach, but all lack the sensitivity and higher titters are needed in eachsample (Mattison and Bidawid 2009). Molecular methods, using the polymerase chainreaction (PCR), reverse-transcription PCR (RT-PCR) and real-time PCR or real-timeRT-PCR, have become the most commonly applied for the detection of viruses in foodand environmental samples (Mattison and Bidawid 2009; Bosch et al. 2011). Theinability to differentiate between infectious and noninfectious viruses whilst using PCRis one of the major limitations. Integrated systems based on the molecular detection ofviruses after cell culture infection are the most promising techniques to overcome thelimitation (Rodriguez et al. 2009). In addition, many authors have reported that thenumber of infectious viruses did not correlate with the number of genomes detected byreal-time RT-PCR in water samples (Baert et al. 2008; Butot et al. 2008, 2009).Recently, meta-genomic sequencing has been used to define the diversity of viral com-munities in reclaimed water and portable water (Rosario et al. 2009), in faecal samplesfrom diarrhoea patients and in various aquatic samples (Williamson et al. 2008). Bothknown viruses and novel viruses were detected by sequencing from each sample library.As sequencing costs diminish and efficiencies improve, mass sequencing could becomea powerful diagnostic tool.

Adenovirus (AdV)

HAdVs are members of the genus Mastadenovirus in the Adenoviridae family, whichcomprises 51 serotypes classified in six species (A–F) (Okoh et al. 2010). They havedouble-stranded linear DNA and a non-enveloped icosahedral shell that has fibre-likeprojections from each of its 12 vertices (Stewart et al. 1993). AdVs species B (Ad3,Ad7 and Ad21), species C (Ad1, Ad2, Ad5 and Ad6) and species E (Ad4) are responsi-ble for 5–10% of childhood respiratory diseases and conjunctivitis (Wold and Horwitz2007). AdV species F (Ad 40/41) has been recognised as an agent of 5–20% of acute

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GE cases among infants and young children (Haramoto et al. 2010), which can beattributed to consumption of faecal contaminated water and food (Chapron et al. 2000;Dongdem et al 2009; Okoh et al 2010). It is estimated that more than 90% of thehuman population is seropositive for one or more serotypes of AdVs (Fong et al.2010). Pathogenicity differs according to species and serotype, and organ specificityand disease patterns appear to be serotype dependent (Madisch et al. 2006; Larranagaet al. 2007; Wold and Horwitz 2007).

HAdVs are excreted in large numbers in human faeces and are known to occur insewage, raw water sources and treated drinking water supplies worldwide (Dongdemet al 2009; Okoh et al 2010). HAdVs are present at a higher frequency in sewage thanother enteric viruses (Pina et al. 1998) and are excreted in high concentrations frominfected patients (up to 1011 viral particles per gram of faeces). Transmission routes ofAdV infection include faecal–oral, oral–oral and hand–eye contact transmission, as wellas indirect transfer through contact with contaminated surfaces or shared utensils andinhalation of aerosols (Boone and Gerba 2007; WHO 2004). AdVs have been linked torespiratory outbreaks in various settings, including military camps (Chmielewicz et al.2005; Kajon et al. 2007), hospitals (Hatherill et al. 2004), day care centres and schools(Fong and Lipp 2005). AdV resistance to purification and disinfection processes(i.e. Chlorine and UV inactivation) and the virus’s long perseverance in the environmenthave increased the importance of monitoring AdVs from water (Thompson et al. 2003;Jiang 2006). The increased UV resistance showed by AdVs may be related with thedouble-stranded nature of their DNA genome, which if damaged, may be repaired bythe host cell DNA-repair mechanisms (Jiang 2006).

AdV identification is generally based on virus isolation in cell culture, followed byantibody or antigen detection and visualisation by EM (Fong and Lipp 2005). In thepast decade, the progression of molecular technologies, especially the application ofPCR methods, has enhanced the speed and sensitivity of AdV detection in water sam-ples drastically (van Heerden et al. 2003, 2004; van Heerden, Ehlers, & Grabow 2005).HAdVs have previously been detected in environmental samples by PCR-based tech-niques (Bofill-Mas et al. 2006, 2010; Albinana-Gimenez et al. 2009). Although quanti-tative real-time PCR methods for the quantification of some HAdV serotypes in diverseenvironmental samples worldwide have been described (Bofill-Mas et al. 2010; Donget al. 2010; Haramoto et al. 2010), to our knowledge, quantitative data on the occur-rence of HAdV in South African recreational waters are still in its infancy (van Heerdenet al. 2003; van Heerden, Ehlers, & Grabow 2005). AdVs, which have a high occur-rence in water, have been recommended as candidates as indicator organisms for viralpathogens because they fit most criteria for an ideal indicator (Griffin et al. 2003; Fongand Lipp 2005; Katayama et al. 2008). AdVs have been detected in surface water inGermany (Pusch et al. 2005), southern California (Jiang and Chu 2004) and in sewageand polluted river and dam water, as well as treated drinking water in Pretoria (vanHeerden, Ehlers, & Grabow 2005).

Enterovirus (EV)

The waterborne EVs group fits into the Picornaviridae family, which consist of non-enveloped virus particles containing a 7500-nucleotide single-stranded positive-senseRNA genome protected by an icosahedral capsid (Nasri et al. 2007). EVs aresmall RNA viruses with sizes ranging from 22 to 30 nm in diameter (Friedman-Huffman& Rose 1998). Human EVs are sub-classified into polioviruses (PV, serotypes 1–3),

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coxsackieviruses group A (CAV, serotypes 1–22 and 24), coxsackieviruses group B(CBV, serotypes 1–6, echoviruses (ECV, serotypes 1–7, 9, 11–27, and 29–33) and EVs68–71 (EVs, 4 serotypes) (Ehlers et al. 2005). Molecular techniques of EV typing arebecoming progressively accessible, thus allowing new EVs to be continually identified,with EVs 79–101 having been recently described (Oberste et al. 2000).

Carriers of EVs include raw sewage, sewage sediments, rivers receiving sewage, aswell as treated sewage (Kocwa-Haluch 2001). EVs are regularly found in several watertypes including natural water, groundwaters, river waters, coastal marine waters, aero-sols emitted from sewage treatment plants and from solid waste landfills, soils andinsufficiently treated drinking water (Kocwa-Haluch 2001; Vivier et al. 2004). Surfaceand groundwaters are used as a supply of domestic drinking water throughout most ofthe world, as well as for leisure and recreational activities; this often leads to the unin-tended ingestion of microbiologically contaminated water (Melnick et al. 1976). Jean(1999) stated that the severity of the EV outbreak in Taiwan in 1998 might in part havebeen related to the contamination of soil and groundwater. Humans are the only knownreservoir of EVs. EVs survive in human faeces for a long time and through contact theycontaminate hands, utensils, food and water. EVs are highly resistant to disinfection,tolerant to residual chlorine from sewage treatment (Wang et al. 2005) and a wide rangeof salinities (Skraber et al. 2004), enabling their survival in environmental waters. MostEVs are readily inactivated at 42 °C, although some sulfhydryl reducing agents andmagnesium cations can stabilize these viruses, so that they are relatively stable at 50 °C(Wang et al. 2005).

Enteroviral diseases occur most frequently in summer and early autumn. EVsinclude more than 70 distinct serotypes of human pathogens and are known to be themain causative agent (>85%) of aseptic meningitis (Lee and Kim 2002; Lee et al. 2004;Pang et al. 2012). Nonetheless, vaccinations do not exist for many serotypes, with theexception of the PV, so the prevention of diseases caused by these viruses is very diffi-cult (Lee and Kim 2002). One of the most typical EV diseases is poliomyelitis. It isalmost invariably caused by one of the three PV serotypes. PVs may also cause asepticmeningitis or non-specific minor illness (Hyypia et al. 1997). Coxsackieviruses, besideother illnesses, are most often connected with human heart diseases. There are novaccines or antiviral drugs currently available for prevention or treatment of diseasescaused by coxsackieviruses (Kocwa-Haluch 2001).

The conventional diagnostic technique for EVs is propagation in cell culturefollowed by neutralisation to confirm the serotype, which is time-consuming(Kocwa-Haluch 2001). Furthermore, several EVs replicate poorly in cell cultures (Wonget al. 1999; Oberste et al. 2000). Molecular-based assays such as PCR, RT-PCR andreal-time RT-PCR have been shown to be more advantageous than cell culture-basedassays for the detection of enteric viruses in faecally contaminated water (Denis-Mizeet al. 2004; Greening and Hewitt 2008; Miagostovich et al. 2008). Certain areas of the5’ non-coding region of the EV genome are highly conserved among all serotypes.Primers binding to these areas can be used to amplify sequences common to most EVs.Thus, the detection of the EV genome by RT-PCR is a valuable alternative to cell cul-turing for evaluating the virological status of the water environment (Gantzer et al.1998). A multiplex real-time hybridization probe RT-PCR for detection of EV 71 andCoxsackievirus A16 has been reported (Kocwa-Haluch 2001). The results showed highspecificity and sensitivity in detecting EV71 or CV-A16 from 67 clinical specimens,and no other EV serotype was detected. EVs were detected in river water samplestested in Brazil (Miagostovich et al. 2008), France (Hot et al. 2003), Germany (Pusch

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et al. 2005), Italy (Grassi et al. 2010), Kenya (Kiulial et al. 2010) and the Netherlands(Rutjes et al. 2009). The presence of EVs (predominantly coxsackie B viruses) was alsodetected in various types of water samples (Vivier et al. 2001; Ehlers et al. 2005),including treated drinking water samples (Vivier et al. 2004; Ehlers et al. 2005). HumanEVs have been included in the European Union guidelines governing water quality as aparameter for assessing the degree of viral pollution of a water body (Kocwa-Haluch2001).

Hepatitis A virus (HAV)

Six types of hepatitis viruses have been identified (A, B, C, D, E and G), but only twotypes, HAV and hepatitis E virus (HEV), appear to be transmitted via the faecal–oralroute and consequently linked to waterborne transmission (Taylor et al. 1995; Hunter1997). HAV is a small (27 nm in diameter), icosahedral, non-enveloped, single-stranded,positive-sense RNA virus belonging to the family Picornaviridae (Hollinger and Emer-son 2007). HAV is prevalent to South Africa with epidemiological features of both thedeveloped and the developing countries being present (Schwab et al. 1995). In highdensity, low socioeconomic and predominantly black African communities where sanita-tion is insufficient, the infection is acquired sub-clinically in early childhood wherenearly 100% seropositivity by school going children acquire HAV immunity before theage of ten years, whilst in the higher socioeconomic and predominantly white commu-nity, more clinical infections are noted, and immunity rises to about 50–70% in adults(Taylor et al. 2001).

The two biotypes of HAV, that is, human HAV and simian HAV, are the only mem-bers of the genus Hepatovirus (Hunter 1997). HAV strains isolated from around theworld constitute a single serotype and are divided into six genotypes based on phyloge-netic analysis of nucleotide sequences in the VP1/2A region (Lu et al. 2004). Many ofthe human HAV strains studied belong to genotypes I or III (Lu et al. 2004). Themajority of HAVs (80%) belong to genotype I (Vaidya et al. 2002). There is only oneserotype with infection conferring lifelong immunity (Hollinger and Emerson 2007).Patients infected with HAV may excrete up to 105 to 1011 virus particles per gram ofstool (Koopmans et al. 2002). Studies have indicated that faecal excretion of HAV maybe prolonged in HIV-infected individuals, thus serving as an added reservoir of infec-tion (Koopmans et a. 2002). Food or waters can be contaminated with HAV directly byfaecal matter and vomitus or indirectly by exposure to contaminated surfaces (Lam-houjeb et al. 2008; Kovać et al. 2009). Polluted drinking water has been implicated inoutbreaks of HAV (Hunter 1997), and recreational exposure to faecally contaminatedwater has unequivocally been associated with outbreaks of HAV (Hunter 1997), withthe risk of infection increasing with increased immersion in contaminated water (Tayloret al. 1995; Gammie and Wyn-Jones 1997). HAV has also been found in surface riverwater, ground or subsurface water and dam (impoundment) water used for recreational,irrigational and domestic purposes in South Africa (Taylor et al. 2001). These waterresources are used by the non-immune higher “privileged” socioeconomic communitiesfor recreational activities, whilst the predominantly immune lower “poor” socioeco-nomic population uses the same water for domestic, irrigation and recreational purposes(Hunter 1997; Gerba 2000; Taylor et al. 2001). HAV is primarily spread by the faecal–oral route with person-to-person contact being the most significant route of infection.Maximal faecal excretion of HAV transpires two to three weeks prior to the onset ofclinical symptoms and remains infectious for three to four weeks after the alanine

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aminotransferase levels peak (Polish et al. 1999), facilitating the spread of the virus.The infectious dose of HAV is unknown. The infectious dose is presumed to be of theorder of 10–100 virions (Venter et al. 2007), although Grabow (1997) suggested thatone virion can cause infection. These studies imply that even low levels of faecal pollu-tion could pose a threat of infection (Venter et al. 2007).

HAV’s stability against chemical and physical disinfection plays a key role in itspersistence and spread in the environment (Venter et al. 2007). HAV is resistant toconcentrations of free residual chlorine of 0.5–1.5mg/l for 1 h, and exposure to2–2.5mg/l for at least 15min is recommended to inactivate any infectious HAV (Fein-stone and Gust 2002). The virus can withstand temperatures of 60–80 °C for a mini-mum of 1 h (Koopmans et al. 2002), low relative humidity (�25% for 7 days) (Mbithiet al. 1991) and pH values as low as pH 1 for 2 h at room temperature and retains itsinfectivity for up to 5 h, which explains how HAV can pass through the human or pri-mate stomach (Feinstone and Gust 2002). HAV has been shown to survive for monthsin experimentally contaminated fresh water, surface waters, seawater, marine sediments,wastewater, soils and oysters (Hollinger and Emerson 2007) and depending on condi-tions, can be stable in the environment for months (CDC 1988). However, it has beenshown that HAV can be inactivated by UV radiation, autoclaving, formalin, iodine orchlorine, with specific thresholds for duration and intensity (Hollinger and Ticehurst1996).

HAV is not readily propagated in conventional cell cultures and the detection limitof routine diagnostic procedures such as EM and enzyme immunoassays (EIAs) isabout 105–106 viral particles ml�1 of test suspension (Taylor et al. 2001). TheRT–PCR has successfully been applied for the detection of HAV in sludge and watersamples (Taylor et al. 1997), and shellfish (Goswami et al. 1993), seawater (Myintet al. 1994) and environmental water samples (Marx et al. 1998). HAV was detected inthe river and the dam water samples in South Africa where microbiological indicatorsof faecal pollution were absent (Taylor et al. 2001). Data concerning the burden ofHAV infection and disease in South Africa is limited, and consequently, the contributionof treated and untreated drinking water, and recreational water to the burden of HAVinfection in South Africa is unknown (Venter et al. 2007).

Norovirus (NoV)

Norwalk-like viruses (NLVs), now called NoVs, are a group of non-cultivable, geneti-cally diverse single-stranded RNA viruses which form the genus NoV within the Calici-viridae family (Green et al. 2001). The human NoVs (HNoVs) can be separated intotwo groups, the NLVs and Sapporo-like viruses (Atmar and Estes 2001). These virusesare similar in size and morphology and exhibit nucleotide sequence homology but areantigenically distinct (Atmar and Estes 2001). NLVs are responsible for the majority ofoutbreaks of acute GE in patients of all age groups in industrialised countries (Meadet al. 1999; Frankhauser et al. 2002). NoVs demonstrate high genetic diversity and arecurrently divided into five genetically discrete genogroups I (GI) to V (GV) on the basisof sequence comparison of the RNA polymerase and capsid region of the genome(Phan et al. 2007). Three groups GI, GII and GIV are known to infect humans (Loganet al. 2007). Within the genogroups, NoV strains can be further separated into geneticclusters, or genotypes, with genogroup II/genotype4 (GII/4, Bristol/Lordsdale group)virus strains being the most leading worldwide and are endemic in hospitals and long-term care facilities (Noel et al. 1999). Investigations across Europe have recognised

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NoV GII/4 as the predominant viral strains (Lindell et al. 2005), with reports ofepidemic spread of GII/4 NoV variants (Siebenga et al. 2007).

Outbreaks of NoV have been caused by contaminated food and/or drinking water,person-to-person virus transmission and airborne droplets of infected vomitus (Meadet al. 1999; McIver et al. 2001; Koopmans et al. 2002; Laverick et al. 2004). In 2002,84% of outbreaks of infectious intestinal disease in Ireland were either confirmed orsuspected to be NoV (NDSC 2003). Of these outbreaks, 70% occurred in hospitals, andother healthcare settings placing an enormous burden on the healthcare system (Loganet al. 2007). HNoVs are shed in faeces of infected patients at a high concentration; thus,the faecal–oral route via contaminated food or water is a main mode of its transmission(Green et al. 2001). Contaminated water poses a particularly serious health risk sinceresults from human volunteer studies indicate that the minimum infectious dose of NoVmay be as low as 10 to 100 PCR units (McIver et al. 2001). Waterborne outbreaks havebeen caused by contaminated surface water, groundwater, drinking water and mineralwater (Beuret et al. 2002; Abbaszadegan et al. 2003). NoVs are environmentally stable,able to endure both freezing and heating (although not thorough cooking), are resistantto several chemical disinfectants and can persist on surfaces for up to 2weeks (Hallet al. 2011). Two studies have confirmed the presence of RNA specific for NoV in dif-ferent brands of European mineral water, thus indicating that bottled water could alsobe an important source of the viral infection (Beuret et al. 2002; Kovać et al. 2009).NoVs have now become the leading cause of endemic diarrhoeal disease across all agegroups (Hall et al. 2011), the leading cause of foodborne disease (Scallan et al. 2011),and the cause of half of all GE outbreaks worldwide (Patel et al. 2009). In the UnitedStates alone, NoVs are responsible for an estimated 21 million cases of acute GE annu-ally, including >70,000 hospitalizations and nearly 800 deaths (Hall et al. 2011, 2012;Lopman et al. 2011, 2012). Whilst in developing countries, where the greatest burdenof diarrhoeal disease occurs, NoVs have been estimated to cause up to 200,000 deathseach year in children <5 years of age (Patel et al. 2008). In waterborne outbreaks, ahigh proportion of the population can be affected, leading to several to hundreds ofcases of GE, followed by secondary spread and resulting in significant economicimpact. NoV outbreaks are difficult to control and present a major public health chal-lenge; thus, rapid diagnosis can be critical for the control of outbreaks. GI NoV strainshave been detected repeatedly in sewage, effluent and surface waters (Myrmel et al.2006; Katayama et al. 2008), which adds to the view that many NoV infections aresymptomless, with GI viruses being under-represented among those found in clinicalcases.

HNoVs cannot be cultivated in traditional cell culture or in animal models (Duizeret al. 2004). Traditional methods of EM and enzyme immunosorbent assay, which wereused in clinical diagnosis, lack adequate sensitivity for analysis of environmental sam-ples (Greening and Hewitt 2008). However, progresses in molecular techniques in thelast two decades have facilitated their detection in clinical and environmental samples(Atmar and Estes 2001). RT-PCR is currently the most extensively used assay for detec-tion of NoVs in environmental water (Karim and LeChevallier 2004; Greening andHewitt 2008), in the Netherlands (Lodder and de Roda Husman 2005) and in Germany(Pusch et al. 2005). Furthermore, this method coupled with nucleotide sequencingtechniques is able to assemble valuable information on the NoV genotypes occurring inthe environment, thus providing epidemiological information of NoV infections in thecommunity. The RT-PCR primers that target the viral RdRp gene in open reading frame1 (ORF1) or capsid gene in ORF2 have been designed to detect and genotype various

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NoV strains (Kojima et al. 2002; Vinje et al. 2004). The RT-PCR format offers the abil-ity to detect lower levels of the virus, as well as a broad spectrum of viruses from bothgenogroup I and genogroup II, thus for these reasons, RT-PCR testing for NoVs can bevaluable in the evaluation of outbreaks (Kojima et al. 2002).

Rotavirus (RV)

RV are large (70 nm) non-enveloped icosahedral viruses that fit into the family Reoviri-dae (Weisberg 2007). RV particles consist of a triple-layered protein capsid surrounding11 segments of a double-stranded RNA genome (Dennehy 2007). The RV genus hasbeen divided into groups, subgroups and serotypes based on viral capsid proteins. RVare classified into at least five groups (A to E), and there are possibly two more groups(F and G) based on the reactivities of the VP6 middle layer protein (Estes 2001). GroupA RV are generally associated with human infections (Kapikian et al. 2001). RV havebeen established as the main cause of acute GE in young children worldwide (Kapikianand Chanock 1996). RV are accountable for an estimated 500,000 deaths each year indeveloping countries (Parashar et al. 2003). An estimated 110,000–150,000 childrenyounger than 5 years of age die annually on the African continent due to RV infection(Molbak et al. 2000; Parashar et al. 2003). Clinical symptoms of RV infection includediarrhoea, fever and vomiting. RV have been estimated to cause 25–35% of all cases ofsevere diarrhoeal illness resulting in a significant economic impact on society in termsof direct medical costs, loss of working hours, quality of life and mortality (Glass et al.1999, 2005). RVs have also been shown to be an imperative cause of sporadic and epi-demic (Taylor et al. 1997; Sebata and Steele 2001; Steele et al. 2004) paediatric GE inSouth Africa.

After replication in the gastrointestinal tract, RVs are excreted in high numbers inthe faeces of infected individuals and may enter various sources of water, such as sew-age (Dubois et al. 1997; Baggi and Peduzzi 2000), river water (Baggi and Peduzzi2000), groundwater (Abbaszadegan et al. 2003) and even treated drinking water(Gratacap-Cavallier et al. 2000). The stability of RV in environmental waters and theirresistance to water treatment may facilitate transmission to humans via the faecal–oralroute (Ansari et al. 1991). RVs were detected in surface water in Germany (Pusch et al.2005), Italy (Grassi et al. 2010) and the Netherlands (Rutjes et al. 2009). Group A RVshave been detected in untreated and treated drinking water samples in southern Africa(van Zyl et al. 2004, 2006). van Zyl et al. (2006) also demonstrated the similaritybetween the environmental types to those clinical RV strains in patients. Frequently, thelaboratory protocols used regularly to detect group A RV, such as EM, EIA and poly-acrylamide gel electrophoresis, are not sensitive enough to detect the virus in concentra-tions represented by less than 1000 RNA molecules (Schwarz et al. 2002). Sensitivemolecular assays and group- and type-specific reverse transcriptase PCR (RT-PCR)techniques have also been used to detect and genotype RVs from contaminated watersources (Hopkins et al. 1984; van Zyl et al. 2006). In addition, a number of RT-PCRprotocols have been developed for the specific detection of RV of various species (Moriet al. 2001). In South Africa, infections related to RV account for approximately 25%of all diarrhoeal hospital cases yearly, with 83% of infections occurring in infants>12months of age (Steele et al. 2004). Results have shown that 95% of the RV-positiveenvironmental water samples is as follows: the Group A RV G type in Kenya (Kiulialet al. 2010) and in South Africa (van Zyl et al. 2006), as compared to the typing rate

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of 70 and 43.6% reported for Egypt and Spain, respectively (Villena et al. 2003), and20% reported for Brazil (Miagostovich et al. 2008).

Astrovirus (AstV)

AstVs are small, non-enveloped icosahedral positive-sense, single-stranded RNA virusesthat are 28–30 nm in diameter with a smooth margin and a star-like EM appearance(Matsui and Greenberg 1996). Seven serotypes of human astrovirus (HAstV) have beenidentified (Matsui and Greenberg 1996). Outbreaks of AstV-associated GE are beingreported with increasing frequency (Oishi et al. 1994; Chapron et al. 2000).

AstVs are transmitted via the faecal–oral route, and outbreaks have been linked withthe intake of water from streams or raw sewages (Cubitt 1991; Liu et al. 2006; Meleget al. 2006). The faecal–oral route is the predominant mode of transmission of AstVand has been established by numerous volunteer studies (Martin 1992; Glass et al.1996; Chapron et al. 2000; Taylor et al. 2001). In 1979, and colleagues studied a filtratefrom a child with mild GE by EM and determined that it contained a large number ofAstV particles. In a previous study, 70% of the environmental samples analysed fromareas in South Africa were positive for HAstVs (Marx et al. 1998). The prevalence ofHAstV in South African patients with GE was found to be between 5.1 and 7% (Marxet al. 1998). HAstV infections occur generally in young children and the elderly (Glasset al. 1996), although individuals of all age groups may be affected (Oishi et al. 1994).In one study, HAstV was found to be the second most important virus associated withGE in hospitalised patients next to RV (Marx et al. 1998).

There are little data on the presence of HAstV in water sources used for domesticand recreational purposes in SA (Taylor et al. 1995; Abad et al. 1997). AstVs weredetected in sewage sources from the Tshwane (Pretoria) Metropolitan Area in SouthAfrica, and these viruses are closely related to clinical AstV isolates based on phyloge-netic analyses (Nadan et al. 2003). In temperate regions, most AstV infections aredetected in the winter whilst in tropical climates, and infections are noted during therainy season (Matsui and Greenberg 1996). HAstV are not readily proliferated in con-ventional cell cultures (Matsui and Greenberg 1996), and the detection limit of routinediagnostic procedures such as EM and EIAs is about 105–106 viral particles ml�1 oftest suspension (Glass et al. 1996). AstVs have been detected in environmental samplesby RT-PCR which has proven to be more sensitive than EM and EIA (Chapron et al.2000; Le Cann et al. 2004).

Conclusion

Waterborne diseases, especially those of viral origin, have a major public health andsocioeconomic impact and are the most important concern of water quality. Entericviruses influenced by faecal contamination are the key pathogens frequently isolatedfrom surface waters and have been associated with many waterborne outbreaks. Variousranges of enteric virus genera and species have been found to colonise the gastrointesti-nal tracts of humans producing a series of clinical manifestations and erratic epidemio-logical features. From a public health perspective, the most important of these are theRV, NoVs, EVs as well as HAV and HAE viruses. All of the identified human patho-genic viruses that pose a significant public health risk in the water environment aretransmitted via the faecal–oral route. Viral monitoring and isolation techniques, althoughnot perfected for all water types, have been developed to isolate and detect viruses

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present in water. New and improved molecular techniques such as real-time PCR andintegrated cell-culture PCR have also been developed for the quantitation of viruses inwater and shellfish. Given the paucity of viral guidelines in water, it is now time toconsider the establishment of virus standards for potable and non-potable waters. Suchguidelines are absolutely necessary for enhanced wastewater effluent treatment and forthe planned use of renovated wastewater for domestic and industrial usage. The signifi-cance of enteric viruses as causative agents of crucial human diseases cannot be overes-timated, and hence, continuous monitoring of water environments cannot be overrated.

AcknowledgementsThe authors would like to thank the Water Research Commission of South Africa and theNational Research Foundation for funding.

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