Insights into Gastrointestinal Virome: Etiology and Public Exposure

Recycled wastewater is widely used owing to the potential shortage of water resources for drinking purposes, recreational activities, and irrigation. However, gut microbiomes of both human beings and animals negatively affect this water quality. Wastewater contamination is continuously monitored, using fecal contamination indicators or microbial source tracking approaches, to oppose arising enteric infections. Viral gastroenteritis is considered a principal manifestation of waterborne pathogenic virome-mediated infections, which are mainly transmitted via the fecal-oral route. Furthermore, acquired enteric viromes are the common cause of infantile acute diarrhea. Moreover, public exposure to wastewater via wastewater discharge or treated wastewater reuse has led to a significant surge of public health concerns. In this review, we discussed the etiology of waterborne enteric viromes, notably gastrointestinal virus infections, and public exposure to municipal wastewater. Conclusively, the early human virome is affected mainly by birth mode, dietary behavior, and maternal health, and could provide a signature of disease incidence, however, more virome diversification is acquired in adulthood. A multi-phase treatment approach offered an effective means for the elimination of wastewater reuse mediated public risks. The insights highlighted in this paper offer essential information for defining probable etiologies and assessing risks related to exposure to discharged or reused wastewater.


Introduction
Virome diversity accounted for approximately 1031 members worldwide, including bacteriophages, as the major division according to a ten-fold diversity evaluation compared to their bacterial hosts' diversity [1]. Likewise, phage community dominates the early human enteric virome, along with the bacterial communities' expansion acquired by maternal-mediated vertical transmission and after weaning [2,3]. On the other hand, enteric viruses in infants are minimally acquired from their mothers [4], however, the enteric viromes mostly persist after the first two years [2,5]. Several enteric viruses could transfer to infants, and even children, via maternal transmission, through direct exposure, or through the use of contaminated water, such as hepatitis E virus (HEV) and hepatitis A virus (HAV) causing gastroenteritis or, in severe cases, fulminant hepatitis [6][7][8].
However, adenovirus, rotavirus, norovirus, hepatitis A virus, and astrovirus represent the most commonly acquired enteric viruses and contribute to virome shaping in its early phases [6,[9][10][11][12]. Moreover, these viruses are of high persistence in various water environments. For example, noroviruses can survive in ground water for 1266 days at 25 • C with 1.76 log 10 reduction [13]. However, adenoviruses can last for 36 and 132 days at 20 • C and 4 • C, respectively, and are associated with a 1 log 10 reduction [14]. Rotaviruses can also persist in fresh water and drinking water for 10 [15] and 64 [16] days, respectively, at 20 • C with 2 log 10 reduction.

Adenovirus
Adenoviruses are members of the family Adenoviridae and the genus Mastadenovirus, comprising of more than 80 human serotypes [57]. Human adenoviruses are non-enveloped icosahedral particles with a double-stranded linear DNA genome of~34-36 kb [58]. They are currently grouped into seven human adenovirus species (A-G), alongside novel adenovirus types that are continuously emerging [59,60]. Virus types were identified in cross-neutralization assays as serotypes up to type 51, however a genotype designation was used for the more recent types based upon phylogenetic analyses of genes encoding the major capsid proteins [61].
Adenovirus infections can lead to a wide spectrum of clinical symptoms. Gastrointestinal infections are commonly caused by subgroup A, D, and F, while subgroup B is the main cause of infections of the lungs and the urinary tract. Subgroups C and E are, however, mainly related to infections of respiratory tract. Amongst subgroup F, serotypes 40 and 41, with serotype 31 of subgroup A, are mainly associated with gastroenteritis [62]. Adenovirus infections are mostly self-limiting, with the exception of immunocompromised individuals. However, a strain of adenovirus 14 that emerged previously resulted in a fatal respiratory disease in healthy personnel [63].
Human adenoviruses (HAdV) are specific to humans even though adenoviruses infect a range of animals. In domestic sewage, HAdV existed in notably high concentrations and their seasonal variability was insignificant [64][65][66]. As with most enteric viruses, adenoviruses are more persistent in various water environments, including lakes, irrigation water, and treated sewage (Table 1), than the currently used fecal indicator bacteria [67,68]. For instance, adenovirus was detected in rivers (18-100%), recreational water (40-93.1%), raw sewage water (0.4-100%), and treated effluents (25-100%, except for that reported in earlier study conducted in Tunisia) as shown in Table 1. Moreover, adenovirus was of the highest concentrations, estimated at 9.8 × 10 8 GC/mL, in treated water influents and in treated effluents, at 4.9 × 10 8 GC/mL, in Italy. HAdV was of the highest frequency (100%) in raw sewage water in all countries, except for the U.K. and Italy, wherein the encountered frequency was 90% and 96% in the same source, respectively. However, the highest HAdV concentration was obtained from lagoons and beaches in Brazil (10 9 GC/L). Furthermore, adenoviruses are highly resistant to UV light and this significant resistance might be due to the host cell-mediated DNA repair mechanism [69]. In addition, adenoviruses have another mechanism that ameliorates the DNA damage response, mediated by the E4 or F4 protein, which are involved in efficient adenovirus replication [70]. Therefore, adenoviruses were proposed as a virological index for water quality control due to their potential environmental stability [71].   [92] a : mean adenovirus concentration, b : maximum adenovirus concentration, c : maximum detection limit (frequency), GC: genome copy. *: genome equivalent, **: treated water before chlorination, ***: treated water after chlorination, ****: last storage reservoir before treatment of drinking water, (-): not defined.
Rotaviruses are divided into seven serogroups (A-G) [96]. Rotavirus A is considered the principal cause of severe acute gastroenteritis throughout the world and predominantly results in severe acute diarrhea in children [97,98]. Moreover, the incidence of rotavirus diarrhea in developing countries accounts for 0.07 to 0.8 episodes per child annually [99] or at least one diarrheal episode by five years of age [9]. Rotaviruses showed a high prevalence in different water sources (Table 2). This could be owing to its broad tolerance to a wide range of temperatures (−20 • C to 37 • C) and pH levels (3 to 11) without a significant infectivity loss [100][101][102][103][104]. However, rotavirus A prevalence is influenced by seasonal variations, favoring lower temperatures in temperate countries [22,105], unlike the endemic manner of rotavirus in tropical countries [106]. In terms of rotavirus frequency, sewage influent of Brazil and, surprisingly, wastewater treatment plant (WWTP) effluents in China recorded the highest frequency, even higher than that reported in Chinese raw water, indicating a Water 2021, 13, 2794 6 of 34 deficiency in the WWTP performance or probable sampling cross-contamination, as shown in Table 2. Moreover, rotaviruses were detected in rivers (18.75-83.33%), raw sewage water (21.2-100%), and treated effluents (5-100%). Furthermore, rotaviruses were detected at significantly high concentrations, up to 1.16 × 10 7 GC/L, in treated water influents and in treated effluents, at levels of 2.8 × 10 6 GC/L, in Brazil and the USA, respectively, as displayed in Table 2. sewage water

Rotavirus
Rotavirus is a double stranded RNA virus composed of 11 segments of a genome size of ~18,550 bp [93]. These segments differ in size from 667 to 3302 nucleotides ( Figure 1). Viral capsid proteins (VP1, VP2, VP3, VP4, VP6, and VP7) are encoded by segments 1, 2, 3, 4, 6, and 9, respectively. The non-structural proteins (NSP1, NSP2, NSP3, NSP4, NSP5, and NSP6) are encoded by segments 5, 8, 7, 10, and 11, respectively. All segments have methylated cap structures at the 5′ end and a 3′UGACC consensus sequence instead of the poly-A tail [94,95]. Rotaviruses are divided into seven serogroups (A-G) [96]. Rotavirus A is considered the principal cause of severe acute gastroenteritis throughout the world and predominantly results in severe acute diarrhea in children [97,98]. Moreover, the incidence of rotavirus diarrhea in developing countries accounts for 0.07 to 0.8 episodes per child annually [99] or at least one diarrheal episode by five years of age [9]. Rotaviruses showed a high prevalence in different water sources ( Table 2). This could be owing to its broad tolerance to a wide range of temperatures (−20 °C to 37 °C) and pH levels (3 to 11) without a significant infectivity loss [100][101][102][103][104]. However, rotavirus A prevalence is influenced by seasonal variations, favoring lower temperatures in temperate countries [22,105], unlike the endemic manner of rotavirus in tropical countries [106]. In terms of rotavirus frequency, sewage influent of Brazil and, surprisingly, wastewater treatment plant (WWTP) effluents in China recorded the highest frequency, even higher than that reported in Chinese raw water, indicating a deficiency in the WWTP performance or probable sampling cross-contamination, as shown in Table 2. Moreover, rotaviruses were detected in rivers The rotavirus outer capsid consists of two distinct neutralization antigens that are responsible for rotavirus attachment and entry, termed VP7 and VP4. They serve for virus classification, with each categorized into a G-genotype (16 genotype) and P-genotype (27 genotype thus far) [98]. Despite the wide spectrum of rotavirus genotypes resulting from G/P combinations, epidemiological studies showed that the most prevalent genotypes are G1P(8), G3P(8), G4P(8), G9P (8), and G2P(4), which cause up to 90% of severe RVA infections worldwide [107,108]. Furthermore, there is no clear relationship between rotavirus genotypes and the severity of disease [109].

Norovirus
Norovirus (NoV) is a member of the family Caliciviridae, with a polyadenylated, positive-sense, single-stranded RNA genome sized~7.5 kb. The ≥40 genotypes are classified into seven genogroups (GI-GVII) [121,122]. NoV infection is considered the most prevalent non-bacterial mediated gastroenteritis, causing~20% of entire gastroenteritis cases worldwide [123], particularly in five years old and younger children [124]. NoV causes~685 million diarrheal episodes [125] and 200,000 deaths per year [126]. Norovirus infection symptoms occurs after an average incubation period of 24 to 48 h, typically involving vomiting, nausea diarrhea, dehydration, fever, and abdominal cramps [127].
Analyses of outbreaks identified GII noroviruses as the most frequently circulating strains causing gastrointestinal infections worldwide [150]. Over the past 20 years, GII.4 became the predominant genotype, resulting in 70-80% of NoV outbreaks in various countries [151]. This genotype is potentially evolving, yielding new pandemic variants, including Grimsby 1995 (or US95_96), Farmington Hills 2002, Hunter 2004, Den Haag 2006b, New Orleans 2009, and Sydney 2012 [152,153]. This strain diversity arises from both genome recombinations and mutational events, since significantly higher non-synonymous changes were observed in comparison with other NoVs, supporting the antigenic drift proposal, although occurring at a higher rate [154,155].
Noroviruses are mainly transmitted via the fecal-oral route, through the ingestion of contaminated food or water, or by oral contact with a contaminated fomite existing in the surrounding environment [156]. Moreover, high rates of secondary infection arise via airborne transmission, although the fomite route is more dominant [157,158]. The biological characteristics of norovirus were extensively studied through human feeding study volunteers [159][160][161][162]. Norovirus inocula as low as 10 viral particles were sufficient to initiate infection [161,163]. This potentially low count-mediated infection is regarded as highly critical when discussing norovirus survival. Strikingly, norovirus was depicted to be of stable infectivity under freezing and thawing conditions [164], although a more recent study showed altered stability upon exposure to three cycles of freezing and thawing [165]. Moreover, it shows a high capacity of survival in a wide spectrum of water bodies (Table 3), as well as thermal resistance, despite being exposure time-restricted to up to 21 min decimal reduction time (time required at a given temperature to perform a log reduction) in the temperature range of 50-72 • C [166,167]. Furthermore, longer exposure time was detected at 50-60 • C, despite irreversible capsid disruption at >65 • C and loss of binding capacity at 72 • C [168]. This high survival capacity can be demonstrated in a norovirus outbreak that occurred in a long-term care facility in which fomite-mediated survival of norovirus resulted in a continuous infection for 14 days following the initial peak of illness [169]. Notably, noroviruses were detected in rivers (0-100%), recreational water (25-50%), raw sewage water (2.8-100%), and treated effluents (1.6-100%), as displayed in Table 3. Furthermore, noroviruses were detected at the highest concentrations, of 7.9 log 10 GC/L, in combined wastewater in the USA (Table 3).

Hepatitis A Virus (HAV)
The hepatitis A virus belongs to the family Picornaviridae, genus Hepatovirus, and is a non-enveloped positive-sense, single-stranded RNA virus of~7.5 kb genome packaged within a 27-32 nm icosahedral capsid [186]. The HAV genome is composed of a single ORF, whose translation occurs by means of a cap-independent mechanism, making the use of the internal ribosome entry site (IRES) located upstream of the genome producing a polyprotein composed of~2230 amino acids [187]. This polyprotein consists of three distinct domains (P1, P2, and P3), which are further processed into 10 mature proteins by the virus-encoded proteinase, 3C pro [186,188]. P1 encodes the four major capsid proteins VP1-VP4. The nonstructural viral proteins are comprised of the polyprotein domains P2 and P3, and also "processed" by 3C pro [189]. HAV displays a high degree of conservation of the antigenic determinants-notably in amino acid sequences of viral capsids-now expanded to include the recently identified HAV-like viruses [190,191]. This could have resulted from negative selection pressures imposed upon any naturally-occurring mutants, producing the observed consensus conservation [192]. Despite the high conservation of HAV, a degree of genomic sequence divergence exists defining the various HAV genotypes and the identity of sub-genogroups [192,193]. Consequently, HAV genotyping is dependent on different regions of its genome used to recognize HAV variants, including the VP1 entire region, notably the VP1 amino terminus, the 168 bp VP1-2A junction, the VP1-2B region, the VP3-2B region, the VP3 carboxy-terminus, and the 5 untranslated region [194,195]. Based on VP1-2A junction region variability (of~15%), seven genotypes of HAV were primarily defined. However, according to the 23.7% variation denoted by the entire VP1 sequence analyses, six HAV genotypes (I-VI) are currently defined, encompassing genotypes 1A, 1B, II, III, IV, V, and VI [196,197]. Genotypes I, II, and III infect humans and are divided into A and B subtypes, however genotypes IV to VI are called simian HAV (SHAV) since they infect non-human primates [196,198]. Amongst human HAV genotypes, subtype IA was found to be the most frequently circulating subtype worldwide [199]. Interestingly, individuals cannot be re-infected by HAV since the presence of a single HAV serotype results in the neutralization of IgG production against HAV, elicited upon vaccination or even natural infection [200].
On the other hand, HAV infections can range in associated severity from asymptomatic to fulminant hepatitis-mediated deaths [201,202]. However, HAV commonly causes selflimiting infections that do not lead to chronic liver disease [200,203]. Moreover, clinical manifestations can increase with age, manifested by jaundice, and unusually high serum aminotransferase levels as the common symptoms, which are exhibited by over 70% of infected adult patients [201,204]. Furthermore, the incubation period of HAV lasts for 15-50 days, with an average of 28 days [205]. A wide range of symptoms occur upon HAV infection, including gastroenteritis, malaise, fever, nausea, anorexia, jaundice, dark urine (genitourinary symptom), and abdominal discomfort [206]. Fulminant hepatitis is considered as a rare complication associated with HAV infections that occurs in less than 1% of infected patients, wherein the highest incidence rates occur in young children and the elderly with reported underlying liver illnesses [205,207]. Nucleotide substitutions at the 5 UTR, P2, and P3 regions of the HAV genome were found to be associated with this fulminant disease [207,208].
HAV is mainly transmitted via the fecal-oral route, as well as through personal contact and exposure to contaminated water/food supplies, whereas transmission routes of the other typically hepatitis-causing viruses, in particular hepatitis B and C, involve contaminated blood or other body fluids via injection, intimate contact, or perinatal period vertical transmission [209]. Remarkably, waterborne HAV outbreaks are uncommon in developed countries owing to proper sanitation procedures, as well as water supply facilities [210]. On the other hand, HAV was found to be of high stability and abundance in the surrounding water environments (Table 4) for long periods, whenever associated with organic matter [207,211]. For instance, HAV was detected to be infectious after more than one year of storage at 4 • C in bottled water, with <1 log reduction owing to concentrations of the added proteins [211,212]. Moreover, HAV shows significant resistance to surprisingly low pH, since it was reported that infectivity remains after treatment at pH 1 for up to 5 h at room temperature, and for 1.5 h at 38 • C and a pH 3 for up to 21 days at 4 • C [213,214]. The environmental stability of HAV, displayed by its low pH as well as heat resistance (60 • C for 1 h), could be due to the inherent molecular stability of the HAV capsid, concurrent with its particular codon usage, along with the unique folding pattern of the VP2 protein [190,215,216]. Moreover, HAV was detected in rivers (1.19-76%), recreational water (0-13.95%), raw sewage water (1.75-100%), and treated effluents (0-64.7%), as shown in Table 2. In addition, HAV was detected at potentially high concentrations, up to 6.0 × 10 6 GC/L, in treated effluents and, at 2.7 × 10 6 GC/L, in raw sewage water in Tunisia.
Ingestion of HAV-contaminated food accounts for 2-7% of all HAV-mediated outbreaks worldwide [217]. Epidemiological investigations provide a potential solution since they have succeeded previously in identifying the source of contamination. For example, a large and persistent food-borne mediated multi-state HAV outbreak occurred in Europe, from 2013 to 2014, which was determined to be due to the ingestion of HAV-contaminated frozen berries. This led to over 1589 cases and 2 deaths [218,219]. Moreover, bivalve molluscan shellfish was reported to have significant HAV levels, showing various prevalence spatially and temporally [220][221][222]. For example, the prevalence of HAV severely declined over the years from 40% to <8%, according to the 20 year-systematic survey conducted on bivalve molluscan shellfish from three estuaries in Spain [220], accompanied by a reduction in HAV cases. However, the reduction in cases could be due to the increasing availability of the HA vaccine, alongside increased surveillance that can rapidly identify contaminated food [217,223].

Astrovirus
Human astroviruses (HAstV) are members of the family Astroviridae, genus Mamastrovirus. They are non-enveloped icosahedral viruses, with a linear positive sense, single-stranded, RNA genome ranging from 6.4-7.9 kb [238]. The genome consists of three ORFs, comprising of ORF1a, ORF1b, and ORF2, which are flanked by 5 and 3 UTRs (85 and 83 nts, respectively) and a 3 poly-A tail (Figure 3). ORF1a and ORF1b encode two functional polyproteins (nsp1a and nsp1ab), encompassing a serine protease and a RNA-dependent RNA-polymerase (RdRp). ORF2 encodes the capsid proteins precursor, translated from a sub-genomic RNA (sgRNA), and comprises of two principal domains: the highly conserved amino (N)-terminus and the hypervariable carboxy (C)terminus [239,240]. In addition, the viral genome encodes genomic linked protein (VPg) that plays a major role in viral infectivity (notably the TEEEY-like tract), the replication of the virus genome, and protein synthesis [241,242]. HAstV was commonly associated with the incidence of acute gastroenteritis in young children, immunocompromised individuals, and the elderly. HAstV is responsible for sporadic non-bacterial diarrheal cases, representing up to 20% and 0.5-15% of related outbreaks [243][244][245]. HAstV is considered the second or third major cause of infantile gastroenteritis after rota-and calciviruses [246]. Nonetheless, regional studies demonstrate a significantly different relative prevalence of HAstV in water resources (Table 5) and clinical settings. For instance, in particular developing countries, 30% of all diarrheal cases HAstV was commonly associated with the incidence of acute gastroenteritis in young children, immunocompromised individuals, and the elderly. HAstV is responsible for sporadic non-bacterial diarrheal cases, representing up to 20% and 0.5-15% of related outbreaks [243][244][245]. HAstV is considered the second or third major cause of infantile gastroenteritis after rota-and calciviruses [246]. Nonetheless, regional studies demonstrate a significantly different relative prevalence of HAstV in water resources (Table 5) and clinical settings. For instance, in particular developing countries, 30% of all diarrheal cases were due to HAstV infection [94,247]. Moreover, HAstV was detected in rivers (8.3-100%), seawater (4-11%), raw sewage water (40.2-100%), and treated effluents (0-84%), as displayed in Table 2. Moreover, HAstV was detected at the highest concentration estimated, 4.3 × 10 7 GC/L, in raw sewage water in Uruguay in comparison to other countries.
Gastroenteritis caused by HAstV is characterized by symptoms involving 2-3 daywatery diarrhea, vomiting, abdominal pain, malaise, and headache [248]. The incubation period is somewhat longer than gastroenteritis caused by other types of virus at an average of 4.5 days [96,249]. HAstV encompasses eight genotypes, HAstV-1 to HAstV-8 [250], while HAstV-1 is the most common genotype identified in both wastewater and stools [251].

Public Exposure to Municipal Wastewater
People are exposed to wastewater by various means (recreational activities, food production, and agriculture). However, the infection risk due to waterborne enteric viruses is dependent on the infectious enteric viruses' prevalence or their infectivity in various water environments that humans interact with (Table 6). For instance, infection risk due to HAdV in drinking water accounted for 10 −4 /year for each person, which was quite similar to astrovirus infection risk detected earlier in surface water in Mexico [87,263]. Moreover, HAdV and EV were found to be of the highest infectivity rate in secondary treated water and even in post-disinfection final effluent confirmed by cytopathic effect detection. However, a lower infectivity rate of HAdV and EV was detected in raw sewage (64%) and dam water (~65%), respectively (Table 6). Furthermore, infection risk was more reduced in surface water (10,000x/180 days and 10,000x/30 days) than in ground water (1000x/'213 days and 10x/30 days) in the case of RV and AstV, respectively. The main exposure routes frequently include recreational activities and surface water drinking. Moreover, shellfish production is regarded as an indirect route of exposure, since molluscs are filter feeders and, consequently, contaminated water pathogens become concentrated and lead to consumer infection [264,265]. Moreover, enteric viruses were detected using nested PCR for entero-, norwalk-like, and hepatitis A viruses and real-time PCR for adenovirus in 50% to 60% of the total mussel samples (18) obtained from a bioremediation mussel farm [266]. Notably, non-enveloped viruses, such as noroviruses and the hepatitis A virus, can survive in the bivalves' tissues and are highly resilient to degradation [267,268]. Moreover, virus particle size was found to determine whether the particle is degradation resistant or susceptible [265]. For example, <200 nm VLPs are typically of higher degradation resistance when compared to bacteria [269]. Human infectious diseases owing to consumption of contaminated filter-feeders and recreational activities in wastewater-polluted coastal waters account for USD 12 billion annually [270]. Moreover, wastewater-mediated irrigation, in particular sprinkler irrigation, generates aerosols that can cause infection upon direct exposure or ingestion of irrigated crops [271]. It is important to note that the RNA of the pandemic SARS-CoV-2 was detected in treated wastewater, representing a critical issue for usage in irrigation [272]. Therefore, SARS-CoV-2 RNA was investigated in various water resources for their incidence frequency ( Figure 4) and prevalence using different genes (e.g., RdRP, S, N1, N2, N3, ORF1ab, and E) as genetic tracers [273][274][275][276][277][278][279][280][281][282][283][284][285]. However, the highest SARS-CoV-2 detection specificity was obtained when RdRP was applied in RT-qPCR and compared to other SARS-CoV-2 genes [286]. Furthermore, detection of SARS-CoV-2 RNA in treated wastewater is not usually associated with SARS-CoV-2 infection risk, as reported in treated sewage of nine WWTPs in Germany [286]. This could be owing to the significantly higher persistence of SARS-CoV-2 RNA than infectious SARS-CoV-2 in water environments. Moreover, the persistence of infectious SARS-CoV-2 in water environments is reliant on many factors, such as water source, temperature, and the initial virus titer. For instance, a T90 of infectious SARS-CoV-2 at room temperature was found 1.7 and 1.5 days in tap water and wastewater, respectively, that extended to 7 days when a higher initial titer (10 5 TCID50/mL) was applied [26]. By comparison, the T90 values declined in wastewater to a total of 15 min and 2 min at 50 and 70 • C, respectively [26]. Toilet flushing and groundwater production render other routes of direct exposure to wastewater [100,287]. ---Cytopathic effect detection and Direct immunofluorescence assay [290] HAdV: human adenovirus, RV: rotavirus, AstV: astrovirus, EV: enterovirus, ♦ : infection risk (IR), including two parameters: infection risk per person and infection risk reduction. Infection risk per person refers to the probability of infection occurrence for each person exposed to various water sources, whereas, infection risk reduction refers to the amount of risk reduction (RA) in case of water treatment (e.g., via disinfection or UV-treatment) or in case of being exposed for a period of time (represented in days) to different water environments. Ұ : Log 10 reduction ¥ : x fold reduction, a: average reduction, IVP:

Discharge of Wastewater
Wastewater is commonly discharged into surface water resources. In addition to the public health concerns, fecal contamination of wastewater can negatively influence water environments essential for fishing, drinking water, and recreation. Enteric viruses are considered the main cause of waterborne illnesses associated with recreational water, including pools, spas, rivers, etc., and can reach waters via the accidental release of feces or body fluids [292]. Moreover, a surge in non-enteric diseases was reported to arise from wastewater contaminated with significant viral contamination [24,25]. Wastewater treatment (WWT) performance guidelines were established for reclamation and reuse (Table  7). These guidelines are concerned with microorganism levels and the degree of treatment, whereas receiving waters risk management mainly depends on fecal indicator bacteria monitoring [293]. Unfortunately, these bacterial indicators cannot meet the full criteria of the ideal water quality indicators [294]. On the other hand, excreted enteric viruses can be detected in wastewater, but a wastewater treatment plant (WWTP) may not completely eliminate viruses in terms of their concentration and infectivity, thus demonstrating a continued water-related health risk [295]. Furthermore, enteric virus presence in water does not necessarily link to the bacterial indicators' detection as Escherichia coli and coliforms [296]. Additionally, bacteriophage survival in water is more similar to human enteric viruses than the presently used bacterial indicators [293]. For instance, cross-assembly phage is currently implemented alongside pepper mild mottle virus (PMMoV) as the microbial source tracks markers simultaneously in this toolbox approach. This is owing to their inclusive distribution, associated with ever higher densities in sewage, than other detected viruses and the fact they follow a similar pattern to enteric viruses prevalence in different water bodies. Consequently, they are efficient as indicators of virus-mediated fecal pollution in lakes, rivers, and recreational waters [297][298][299][300][301]. Thus, traceability procedures provide a mandate to determine fecal contamination sources so that the risk can be assessed to initiate a proper water management to counteract it at its source.   [274,[276][277][278][281][282][283][284][285][286]291]. SW: sewage water, TWW: Treated wastewater, RW: River water, SS: Wastewater sludge samples, Av.: Average frequency, for example, Av. SW: Average frequency of SARS-CoV-2 RNA in SW of countries that involved SW in their evaluation.

Discharge of Wastewater
Wastewater is commonly discharged into surface water resources. In addition to the public health concerns, fecal contamination of wastewater can negatively influence water environments essential for fishing, drinking water, and recreation. Enteric viruses are considered the main cause of waterborne illnesses associated with recreational water, including pools, spas, rivers, etc., and can reach waters via the accidental release of feces or body fluids [292]. Moreover, a surge in non-enteric diseases was reported to arise from wastewater contaminated with significant viral contamination [24,25]. Wastewater treatment (WWT) performance guidelines were established for reclamation and reuse (Table 7). These guidelines are concerned with microorganism levels and the degree of treatment, whereas receiving waters risk management mainly depends on fecal indicator bacteria monitoring [293]. Unfortunately, these bacterial indicators cannot meet the full criteria of the ideal water quality indicators [294]. On the other hand, excreted enteric viruses can be detected in wastewater, but a wastewater treatment plant (WWTP) may not completely eliminate viruses in terms of their concentration and infectivity, thus demonstrating a continued water-related health risk [295]. Furthermore, enteric virus presence in water does not necessarily link to the bacterial indicators' detection as Escherichia coli and coliforms [296]. Additionally, bacteriophage survival in water is more similar to human enteric viruses than the presently used bacterial indicators [293]. For instance, cross-assembly phage is currently implemented alongside pepper mild mottle virus (PMMoV) as the microbial source tracks markers simultaneously in this toolbox approach. This is owing to their inclusive distribution, associated with ever higher densities in sewage, than other detected viruses and the fact they follow a similar pattern to enteric viruses prevalence in different water bodies. Consequently, they are efficient as indicators of virus-mediated fecal pollution in lakes, rivers, and recreational waters [297][298][299][300][301]. Thus, traceability procedures provide a mandate to determine fecal contamination sources so that the risk can be assessed to initiate a proper water management to counteract it at its source. (-): not applicable.

Reuse of Treated Water
The reusing of wastewater is determined by economic factors as it is often either used for the recirculation of organic matter, to act as natural fertilizers, or due to a shortage of water resources [27,28]. For instance, wastewater and greywater (households' wastewater with no fecal contamination) were used for irrigation of agricultural products, as well as indoor activities involving toilet flushing and even for potable use [17][18][19][20][21]. However, intensive treatment measures are required to meet the suggested wastewater reuse guidelines and, in particular, for greywater in which significant coliform loads may exist. However, the performance of treatment procedures relying on coliform elimination may be biased and exaggerated owing to the capability of these bacteria to multiply within the greywater system.
Wastewater reuse is currently a frequent practice in many countries. For example, treated wastewater is utilized in agriculture and landscaping in many countries, including Egypt, Saudi Arabia, Italy, Cyprus, Malta, Spain, and the USA [306][307][308][309][310]. It is, for instance, used in Egypt for the irrigation of sandy soils to raise the organic matter content of soil and improve the capacity of cation exchange [311]. In Saudi Arabia in 2010, 25% of treated wastewater was used to irrigate landscapes in the public parks of a number of cities [312]. In the Netherlands, particularly in Amsterdam, wastewater is regarded as a rich resource of organic matter, including alginic acid, cellulose, bioplastic, biogas, and phosphorus obtainment, that can be recovered and reused [313]. Moreover, wastewater reuse has converted Singapore into a universal hydro-hub via the implementation of novel water technologies that allowed the nation to meet 30% of its water demands, a number that is set to increase to 55% by 2060 [314]. Wastewater reuse usually demands higher standards of treatment, since it may well contain higher pathogen content than greywater [315]. Greywater reuse is, therefore, much easier when separated from wastewater [316]. However, water contamination is possible in all pathways to an extent that necessitates adequate safety measures prior to the establishment of new systems. In this regard, Singapore has approved a potential multi-phase approach to water reuse, involving primary sedimentation, followed by activated sludge and microfiltration, and then ultrafiltration, reverse osmosis and, eventually, disinfection by ultraviolet radiation exposure [314]. This approach can also be highly beneficial to eliminate or significantly reduce public health risks associated with the reuse of various wastewater streams. However, the targets should be well defined and technical solutions and proper assessment tools should be made available to ensure that it meets the recommended guidelines of safe water reuse.
Technical solutions for reuse of treated water includes: (i) the employment of innovative tools and technologies, such as the replacement of old equipment used for water treatment with new technologies, including membrane bioreactor (MBR)-based treatment [317,318], (ii) the establishment of an evaluation approach for determining water cost, that is energetic and equivalence-dependent, alongside treatment strategies [319], and (iii) solving the gap between water supply and water demand via desalination methodologies [320,321], control of runoff water [322], wastewater reuse [323], and cloud seeding [324]. However, the technical solution selection mandates the inclusion of a short costs analysis that depends on the nature of water reuse projects being chargeable or not [325]. For instance, regenerated water use for industrial purposes or course irrigation in the private sector required investment costs that reached AUD >3/m 3 in Australia [326] and up to EUR 736/m 3 produced/day in Spain, with an additional EUR 0.06/m 3 to EUR 0.45/m 3 for operational costs that varied according to the regenerated water uses and the required treatment [327]. On the contrary, regenerated water for water resources restoration and maintenance, the recharging of aquifers, or reduction of treated effluent discharge into essential water bodies is not chargeable [325,328]. Technical solutions for water reuse should be assessed for, in particular, viral load reduction efficacy to avoid any associated health concerns due to direct exposure. The MBR treatment process was found to be of higher efficiency, in terms of both bacterial and virus removals, than the activated sludge process, which results in up to 2 log 10 bacterial load reduction and lacks virus removal capability [329]. For example, adenoviruses and enteroviruses, and even infectious enteroviruses, were 3.7, 1.7, and 2log 10 reduced, respectively, following MBR-based treatment in Saudi Arabia [318].
Nevertheless, such efforts for the reuse of treated water, alongside the offered technical solutions, were opposed by serious limitations raised against irrigation using treated water. The alteration of the texture properties and physicochemical parameters of soil, due to agricultural reuse, led to changed microbiota and biomass [29]. Moreover, probable modifications of soil microbiota could influence soil fertility and subsequent productivity [21]. In addition, organic matter mineralization [330] and nutrients and metals availability [331,332] were affected by altered soil pH caused by irrigation with WWTP effluents. Therefore, reuse of insufficiently treated wastewater, or even raw water, could serve as alternative sources for irrigation, avoiding these risk factors [29]. However, other risk factors may emerge from the high burden of enteric pathogens. The norovirus disease burden due to the consumption of lettuce irrigated with untreated greywater was assessed by QMRA model and revealed an annual disease burden fluctuation above the range of 2 × 10 −8 and 5 × 10 −4 [333].
On the other hand, wastewater reuse is commonly practiced for potable purposes owing to the incidental presence of treated wastewater in a water supply source, termed de facto wastewater reuse. Upstream WWTP discharge was reported by the EPA as influencing drinking water treatment plants (DWTPs), which included 2-16% of upstream wastewater discharges [334]. For instance, DWTPs, containing 50% upstream WWTP discharges, were used to serve over 10 4 U.S. people, as reported in [335]. However, extensive risk assessment studies were concerned with the associated risks due to de facto reuse [30,336,337] and an annual risk of >1 infection/10 5 people was recorded [338]. Moreover, the median norovirus risk per year was the highest in the case of de facto reuse, when compared to other treatment scenarios, to such an extent that de facto reuse scenarios exceeded risk benchmarks (10 −o ). Alarmingly, 1% wastewater effluent was predicted to potentially surge drinking water risks if contributed to the source water [30]. De facto reuse is still applicable globally and de facto reuse is expected to increase in the future due to an increased frequency of water supplies shortages and droughts [339].
The Water Safety Plan (WSP) is receiving increasing attention as a recommended risk management approach for water reuse. The establishment of the WSP approach organized drinking-water-related management practices and assured the applicability of these practices to drinking-water quality management. A WSP encompasses, at least, a system assessment and effective operational monitoring and management to ensure drinking-water safety. Moreover, a WSP integrates various principles of other risk management approaches including, of a particular interest, the multiple-barrier approach and HACCP [340]. HACCP development presented an earlier framework for the improvement of drinking water treatment processes to minimize the probability of waterborne disease incidence [341]. Moreover, repairing and maintaining the drinking water distribution network was highlighted as a means of preventing drinking water contamination. In this regard, the necessity of HACCP implementation was reported in terms of avoiding microbial contamination incidence in drinking water treatment lines [342].

Conclusions
The high persistence of enteric viruses in various water environments enabled their detection in almost all water sources with a significantly higher frequency. However, detection frequency of enteric viruses could vary according to the virus type, geographical location, water source, and the assigned period for sampling, irrelevant to virus concentration. Second, virus detection is not usually associated with virus infectivity, wherein the presence of infectious virus particles indicate infectivity and the resultant infection risk. Moreover, infection risk relies on detection specificity since the virus origin, whether of human origin or not, could determine the course of infection. For example, the RDRP gene of SARS-CoV-2 was found to be of higher specificity than other genes. On the other hand, exposure to wastewater discharge should be monitored and controlled for probable health issues. Consequently, a toolbox approach, implementing both pepper mild mottle virus and cross-assembly phage together, was highly encouraged for the traceability of any possible fecal pollution. Moreover, MBR-based treatment for wastewater reuse was much more efficient in both bacterial and viral burden reduction than activated sludge, which cannot achieve virus removal. A WSP could likewise help in the quality management of de facto wastewater reuse through the employment of HACCP throughout the wastewater treatment strategies. This review article provided the information necessary for decision making in terms of determining the most probable viral etiologies as well as evaluating the resultant risks associated with direct or indirect exposure to both discharged or reused wastewater.