The aquatic ecosystem, including coastal waters, rivers, and lakes, is continuously menaced by the infiltrations and drainage of anthropogenic wastewaters. Consequently, this environmental hazard requires the perpetual and constant implementation of up-to-date monitoring systems to become more comprehensive, perspicuous, and specific to targeted pollutants [1]. Recently, genomic-based tools substituted the conventional burdensome morphological monitoring tools and vastly implemented as a routine tool in cutting-edge research and biotechnologies. However, the application of the modern genomics tools in environmental biomonitoring is so far considered an innovative approach and demands improved knowledge to be applied in aquatic biomonitoring. Until now, environmental genomics was mainly used to screen known morphologically distinguishable bio-indicator taxa [2].

Enteric viruses (EVs) occur naturally in their infective (active) form in aquatic environments and are commonly introduced together with bacterial and parasitic microbes through anthropogenic activities, including agricultural runoff, urban runoff, leaking sewage and septic systems, sewage outfall and vessel wastewater discharge. Being transmitted through the fecal–oral route, EVs replicate usually and primarily in the epithelial cells of the host gastrointestinal (GIT) tract and are secreted in the feces of infected patients in extremely high numbers (10⁵ and 10¹¹ viral particle/gram of stool) [3]. Besides anthropogenic activities, EVs are commonly secreted indirectly into in aquatic groundwater, rivers, aerosols discharged from sewage/wastewater treatment plants, estuarine water, inefficiently treated water, and drinking water receiving untreated contaminated wastewater, and wastewater-contaminated private wells [3].

The clinical complications of EVs-infections are primarily associated to diarrhea and self-limiting gastroenteritis in infected humans, EVs can also trigger more life-threating complicated syndromes including respiratory tract (RT) infections, conjunctivitis, hepatitis, and diseases that have high severity and high fatality rates (e.g., aseptic meningitis, encephalitis, and paralysis) [4]. In addition, some EVs infections are associated with chronic disorders, e.g., insulin-dependent diabetes mellitus (type 1 diabetes) and inflammatory cardiomyopathy (known as myocarditis) [5]. On the other hand, infections with enteric viruses are commonly asymptomatic in domestic animals (e.g., cattle and swine), but can sometimes lead to unpleasant economic losses such as abortion and diseases of the central/peripheral nervous system of the animal (neurological disorders) ^[3][6][3,6].

2. Conventional Viral Detection Tools for Aquatic Biomonitoring

EVs detection in aquatic water is a complex trait because of the high dilution factor of the obtained samples making these viruses sporadically occurring in environmental samples. In addition, environmental samples contain a variety of naturally occurring inhibitory substances that may mask the presence of active viral particles in the samples. Electron microscopy and animal cell culture systems represented, for a long time, the standard method for detecting infectious viruses in water.

2.1. Electron Microscopy

Electron microscopy (EM) was heavily applied during the 19th century to recognize newly emerging viruses. Using EM, the first successful trial to discover the structure of poliovirus was achieved in 1952 ^[7][57], followed by norovirus (Caliciviridae) in 1972 ^[8][58]. The study of virus-host interactions started with the help of EM in the mid-1950s and subsequent virus classification was mainly dependent on the morphological features, revealed through EM examination ^[9][59]. Despite that EM is considered by many researchers to be an old technique, especially with the availability of highly specific and sensitive molecular diagnostics, EM is a fundamental tool to detect the etiological agent of new or unusual outbreaks caused by emerging and potential bioterrorism viruses. Moreover, EM is currently on the forefront in the field of structural virology especially in studying the correlation between viral ultrastructure and the clinical viral diagnoses and pathogenesis. Moreover, in the research area, EM is highly applied in different modalities including electron tomography, immunoelectron microscopy, and cryo-electron microscopy to study the viral fitness, cellular cascades which are involved in virus replication, and viral-replication cycle ^[9][59]. EM/bioinformatics combination offers the transition from 2D imaging to 3D remodeling which enables structural and functional analyses that broaden the knowledge of the spectacular diversity in viral-particle structure and replication cycle. Together with confocal laser scanning (CLS) microscopy, EM allows live imaging of infected and control cells with high-resolution analysis ^[10][60]. One main advantage of using EM over other molecular and serological methods for viral diagnosis is that EM does not require virus-specific materials for recognition. However, EM can only identify a virus through morphology, making it impossible to identify a virus beyond the family level ^[9][59].

2.2. Cell Culture Systems

Since the early 1960s, cell culture systems were established and routinely used for virus isolation and viral disease diagnosis. Therefore, the cell culture approach was described for decades as the “gold standard” for viral diagnosis. However, the cell culture approach for diagnosis is relatively time-consuming and requires considerable technical expertise. Additionally, cell cultures allow only active viruses to grow. Simultaneously, viruses require specific permissive and susceptible cell lines which may be difficult to manage in the case of environmental samples, where a collection of viral and bacterial pathogens is expected to exist together. With the innovation of non-cell culture methods for the rapid identification of viral nucleic acids and antigens, the importance of viral culture for viral diagnosis retreated ^[11][79]. In combination with electron microscopy and immunofluorescence (IF) techniques, the sensitivity of cell culture models has been increasingly improved. Nevertheless, these staining techniques cannot be implemented to definitively characterize all viruses and distinguish their different subtypes/genotypes.

2.3. Immunological Methods

Most immunological methods for EVs rely on antigen detection such as enzyme immunoassay (EIA), latex agglutination test, or immunochromatography technologies. For instance, the latex agglutination reaction (LAT) was developed in the 1980s to detect bacterial toxin A. LAT assays identify the observed clumping ability when a filed sample comprising a specific antigen is mixed with latex particles with a specific antibody coat on their surface, leading to agglutination. The LAT assay is considered one of the more favored, rapid, and easiest methods. Therefore, it is used currently to rapidly provide a fast diagnosis tool for the identification of several EVs under laboratory and non-laboratory settings. Specifically, in different virology laboratories in developing countries, the LAT assay is so far reputedly used to diagnose EVs in specimens such as human EVs (e.g., rotaviruses, noroviruses, astroviruses, and adenoviruses) and animal EVs ^[8][12][58,80]. Being qualitative with limited sensitivity and specificity, and unable distinguish between infective and defective viral particles, the results of these techniques are questioned. The enzyme-linked immunosorbent assay (ELISA) is known to be one of the most commonly used and easy-to-go serological diagnostic assay. Interestingly, numerous modifications and updates of this technique are currently applied and available as commercial kits to detect EVs. For instance, a fully automated and ultrasensitive bioluminescent enzyme immunoassay (BLEIA) was introduced to detect norovirus capsid antigen. This technique is as sensitive as ELISA in the detection of various EVs (reviewed in ^[8][58]). More recently, a workable sensitive sandwich ELISA to detect norovirus genogroup II (NoV-II) was developed as an applicable assay for NoV-II early stage diagnosis with improved sensitivity ^[13][81].

2.4. Biosensors

Biosensors are ready-to-use measurement devices that can sense several environmental biomolecules. These devices are currently applied on large scale for the detection of clinical pathogens including viruses ^[14][82]. More recently, nanotechnology revolutionized the biosensors in terms of device design and performance via developing nanoparticles that improve these sensors affinity, selectivity, and efficacy in detecting these viral pathogens ^[15][83]. Interestingly, biosensors are portable bioanalytical devices which mainly consist of an analyte, receptor, transducer, and signal reader to detect any biochemical interaction ^[14][82]. Biosensors have been used for detection of viruses in water, clinical samples, and food ^[14][82]. Fortunaly, biosensors are improved to enable virus detection in few minuteshour with high sensitivity and specificity. Moreover, the fast detection analyses of environmental samples using biosensors are cheap compared with other molecular methods, making it an ecomomic detection tool. Compared with PCR-based techniques, biosensors are not affected by inhibitors of molecular methods, which are extensively present in the concentrated water samples ^[16][84]. Several approaches for the development of biosensors have been conducted to detect a variety of waterborne viruses including norovirus, rotavirus, coronavirus, and influenza subtypes H3N2, H1N1, and H5N1 viruses ^[14][17][82,85]. Recently, biosensors concepts have been implemented to inaugurate Lab-On-Chip (LOC) or Point-of-Care Testing (POCT) devices to rapidly detect viruses and for viral disease diagnosis such as microfluidic chips that integrate many laboratory functions on a single chip and combining micro-electro-mechanical systems together with microfluidic technology ^[18][86]. These microfluidic devices have been recently advanced, attracted attention, and made its breakthrough in shortening the time and speed for virus detection. This technology can also significantly adapt virus testing for Point-of-Care in home settings. However, microfluidic chips face challenges for virus detection including: (1) collection and sample preparation integration, (2) application of quantitative methods, (3) and capacity for throughput and multiplex during outbreaks.

3. Viral Genetic Tools for Aquatic Biomonitoring

3.1. Polymerase Chain Reaction (PCR), Sequencing and Phylogenetic Analysis

Despite that cell-culture detection methods are considered the gold standard to isolate and further detect the infectious viral particle from aquatic samples; viral genetic-based molecular techniques became an optimum alternative to rapidly detect the virus in the sample within few hours with less cost. Commonly, most virology laboratories develop and implement polymerase chain reaction (PCR) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) as well-established methods to detect DNA- and RNA-waterborne viruses, respectively. Compared with cell culture, both forms of this conventional PCR reactions are useful in detecting EVs in water samples due to their high specificity and sensitivity, especially in the detection of those viruses with low concentration in water samples and also non-culturable viruses ^[19][87]. Unlike cell culture for waterborne viruses, the PCR method underestimates the true level of contamination due to the usage of highly specific primers to capture the viral genome ^[19][87]. For instance, EVs in 4 (8%) out of 50 household wells were detected by PCR method, while no viruses were detected after inoculation of cell cultures ^[20][88]. The main limitations of the PCR technique is its inability to quantify viruses and also that it cannot define whether the detected EVs in the sample is infective or defective. Therefore, to improve the sensitivity, specificity and efficiency of PCR, updates of the PCR technique including integrated cell culture PCR (ICC-PCR), multiplex PCR, nested PCR (and semi-nested), real-time PCR (RT-PCR) (for quantification), digital PCR (dPCR), and droplet digital PCR (ddPCR) were introduced. Nested and semi-nested PCR methods were developed and implemented to augment the sensitivity and specificity of the PCR techniques with an internal primer through two run reactions of PCR. Nested PCR assays for adenoviruses were shown to significantly increase compared with conventional PCR assay with sensitivity limits of 10⁻² adenovirus particle/mL ^[21][89]. Unfortunately, the improved high sensitivity of the nested PCR may be accompanied with a high probability of subsequent contamination when PCR products from the first round of PCR are transferred to second round of nested PCR ^[22][23][90,91]. On the other hand, real-time PCR (rt-PCR) or quantitative PCR (qPCR) is currently the most significant and widely used for virus detection in water in all environmental virology labs using either a SYBR Green or Taqman probe. Unlike the predefined techniques, qPCR is characterized by rapidity, sensitivity, reduction in contamination risk, and ability to provide quantification of viruses in samples. To the last point, the qPCR assay demands a curve of known concentrations of the standard genome. Using rt-PCR in combination with genotype or subtype specific-primers, it can be used for genotyping of waterborne viruses in the same reaction. Therefore, several studies have used rt-PCR to investigate viral outbreak in water and wastewater ^[24][92]. Previous studies have detected various viral species and viral loads of adenoviruses and EVs by rt-PCR/qPCR compared with the predefined methods ^[25][93]. The critical limitations of qPCR are similar to conventional PCR, including the inability to determine the viral infectivity in the samples and the probability of inhibition by inhibitor substances in wastewater samples that may affect qPCR amplification and lead to false negative results. Since the emergence of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) in 2019, the adoption of dPCR in detecting and quantifying viral pathogens in clinical, environmental, and wastewater surveillance samples has been accelerated. Uniquely, dPCR technologies apply Poisson distribution to estimate the most probable number (MPN) of a genetic target based on the endpoint fluorescence in each individual partition, without the need for a calibration curve. Compared with qPCR, well-optimized dPCR assays are capable of low inhibition rates, better sensitivity, less variation at the quantitative limit, and increased accuracy ^[26][94]. Following PCR-based techniques, nucleotide sequencing and phylogenetic analysis are performed to deeply characterize the amplified viral genetic amplicons (PCR products). Molecular genetic analyses of viruses in water and wastewater samples commonly reveal that EVs found in environmental samples harbor a genetically diverse viral genome (quasispecies) ^[27][95]. By sequencing and phylogenetic analysis of the PCR products, the major genotypes of viruses in aquatic and environmental samples are determined ^[28][96]. On the same hand, the identification of various antigenic subtypes and/or multiple types of EVs in a single run is always preferred and economic to solve the complex etiology and diversity posed by different EVs. Therefore, a multiplex RT-qPCR assay was innovated and implemented to detect diverse EVs (e.g., astroviruses, adenoviruses, rotaviruses, sapoviruses, and enteroviruses) ^[8][29][58,97]. Unfortunately, a multiplex RT-qPCR assay detected norovirus ^[30][98].

3.2. Isothermal Nucleic Acid Amplification-Based Assays

Recently, isothermal amplification (IA) methods including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), single primer isothermal amplification (SPIA), and recombinase polymerase amplification (RPA), are developed for detecting genetic materials of contaminating pathogens including viruses from environmental samples using simpler (economic), rapid, specific, and sensitive techniques. However, these techniques are not yet established in most central virology laboratories, especially in developing countries.

3.3. DNA Microarray Technology

DNA microarrays are microscope slides on which thousands of immobilized individual DNA capture fragments are spotted to hybridize with complementary target sequences in the organism of interest. Interestingly, microarrays approaches can detect multiple viruses (≥10,000 pathogens) using pathogen-specific complementary probes to hybridize target sequencing. Probe-genome hybridization is commonly observed using a reporter fluorescence ^[31][106]. Additionally, the inclusion of different “probe” sequences is capable of providing a tool for simultaneous detection of different types and subtypes. To these points, microarrays are potentially considered as a powerful technology for virus detection with a high specificity degree. However, the sensitivity is relatively low and the test cost is high. Therefore, they are unlikely to be implemented shortly in routine biomonitoring of environmental water samples. Experimentally, microarrays were applied in environmental studies and evaluated for detection of EVs in complex environments. Microarrays were shown to provide a great potential as a specific, sensitive, and quantitative detection tool for EVs in environmental samples ^[32][107]. Similarly, microarrays have been applied with PCR for the detection and identification of rotaviruses, norovirus, human coronaviruses in wastewater ^[33][108], norovirus, HAV, rotavirus, adenovirus, astrovirus, and coxsackieviruses A and B ^[34][109]. Moreover, microarrays were applied in viral genotyping to determine the genotyping of norovirus in environmental and tap water. In addition, microarrays contribute to detect viral pathogens in environmental surface water in pandemic situations (e.g., SARS-CoV-2), where microarrays allowed scientists to determine the persistence of active SARS-CoV-2 virus for days ^[35][110]. Although it is a very good technique, its current application is limited due to its high cost. This technique should now be provided attention in view of its reliability and performance and considering its modulation into a cost-effective assay.

3.4. Next Generation Sequencing (NGS) and Metagenomic Technology

In the past decade, metagenomics and NGS have revolutionized the virological sciences including virus discovery and are widely employed by researchers in diagnostics and research laboratories. Both provide a package service for detection and sequence identification of total viral types and subtypes via analysis of total nucleic acids which are occurring in a complex biological matrix. Based on their ultra-high throughput, scalability, and speed, viral metagenomics and NGS offer an optimum alternative approach to the predefined molecular method. Recent advances in NGS technology and metagenomics approaches facilitated the discovery of new viruses and other emerging microorganisms in environmental samples. Unlike bacteria in which metagenomics describe the diversity of the 16S ribosomal RNA, viral metagenomics describe the full or partial genomes of all viruses present in the sample. Since 2002, viral metagenomics technology were used to determine viral species in environmental samples including freshwater, marine sediment, soil, and the human gut ^[19][87]. Traditionally, the viral metagenomic studies relied on standard cloning protocols of viral samples and then sequencing by Sanger technology ^[36][111]. More recently, the innovation and implementation of novel NGS platforms such as pyrosequencing, Ion Torrent, Illuminia, MinION, and ABI/Solid enabled high-throughput sequencing of RNA/DNA amplicons from known and newly emerging viruses in water and facilitated the discovery of these emerging and reemerging pathogens ^[19][87]. In combination with bioinformatics, NGS can translate a large amount of genomic data into extra knowledge regarding viral genomes ^[37][112]. Viral metagenomics application for aquatic biomonitoring of environmental water and wastewater provide an excellent platform to detect unknown waterborne viruses and/or EVs. In a study of sewage samples, researchers identified 21 viral families, including several human DNA/RNA viruses such as Picornaviridae, and Papillomaviridae ^[38][113]. Recently, several viral pathogens were discovered in water by metagenomic technologies in river water samples ^[39][40][114,115]. Despite that NGS is considered a powerful tool, NGS-based metagenomic studies were limited mainly with three major challenges: (1) sample preparation for high-throughput sequencing, (2) contamination, and (3) specialized bioinformatic analysis. Owing to the high data output of these NGS platforms, data processing, and analysis of these projects generally require strong computational infrastructure and technological expertise ^[37][112]. For instance, data analyses include the clustering of millions of viral genomic reads into many different separate genomes ^[41][116], and identifying these assembled genomes can be hampered by miss-annotations and incomplete reference databases ^[42][117]. The sequence analysis and processing tasks are computationally intensive and generally require dedicated bioinformatics expertise. Conclusively, data output from NGS platforms need workflow to be able to be analyzed. Commonly, the majority of NGS workflow consists of up to five different steps including: (1) assess the data quality, (2) filter, (3) mapping reads, (4) and annotation to reference databases. On the other hand, there are several challenges in analyzing viral metagenomes including: (1) high output of sequencing reads; (2) assembly of millions of genomic fragments; (3) annotation of all assembled genomes to reference databases; and (4) metagenomic data interpretation. To sum, this technique should now be provided a attention in view of its reliability and performance, and considering its modulation into a cost effective assay.

3.5. ChIP-Seq Analysis

Chromatin immunoprecipitation (ChIP) followed by NGS sequencing (ChIP-Seq) is not purely directed to viral diagnosis, it is used to define the interaction between the virus proteins including those for enteric viruses and the genomic DNA. ChIP is an essential technique to study viral protein-genome interactions within the infected cell. This technique includes major steps such as cell fixation, sonication, immunoprecipitation, and sequencing of the immunoprecipitated DNA. Using the ChIP-Seq approach, the knowledge about the genome loci interactions in host cell with EVs was clarified ^[43][44][45][118,119,120]. Using specific antibodies against viral DNA and/or RNA-associated antigens, ChIP-Seq analysis can be developed and implemented for diagnosis of viral pathogens. However, this technique is time consuming and needs special training on different approaches such as immunoprecipitation and sequencing of the immunoprecipitated genetic motifs.