Juliana Schons Gularte, Meriane Demoliner, Ana Karolina Antunes Eisen, Micheli Filippi & Fernando Rosado Spilki
1 Introduction
The contamination of aquatic environments by the release of sewage carrying different microbiological pathogens is a public concern throughout the World (Bonadonna, Briancesco, & La Rosa, 2019; Val et al., 2019). Waterborne diseases affect public health, reflecting on over-costs for healthcare systems (Pang et al., 2019). An important number of waterborne illnesses are preventable if better access to safe water supply, hygiene practices and sanitation facilities are available (Bosch, Guix, Sano, & Pintó, 2008).
Viruses are responsible to trigger most of the reported gastrointestinal illness comparing to other pathogens like bacteria and protozoa (Bosch et al., 2008; Pang et al., 2019). Enteric viruses have a wide range of transmissions routes, including person-to-person contact, zoonotic and vehicle transmission (Bosch et al., 2008). They are released in the environment, being transported into the surface and ground waters by runoff and percolation, respectively (Pang et al., 2019).
Many approaches to detect microbiological agents have been developed in recent decades changing the way water research and monitoring are carried out. However, the detection of pathogens in water samples has still some challenges to overcome, like a low concentration of the pathogens in water bodies that imply a large sample volume to be analyzed, as the presence of inhibitors in the samples that can interfere in the results (Bonadonna et al., 2019). In addition to the issues cited, for the detection and characterization of the viruses, the technical challenges of assays and the viral diversity must be highlighted as well (Bosch et al., 2008).
The virological diagnosis is performed by multiple methods when dealing with biological samples, including viral isolation, serology to detect antigen and/or antibody, direct detection of viral particle and molecular analysis (Santos, 2015). Many of these methods are not applicable to the environmental samples and most of them have to be adapted for this purpose. An ideal approach for viral monitoring in waters may have excellent analytical sensitivity, specificity, being reproducible, has cost-effective and easy to perform (Bonadonna et al., 2019). Seeking to find a methodology closer to this ideal standard, the main objective of this review is to describe some of the most used current techniques to the detection of viruses in environmental samples, especially water samples, showing the advantages and challenges in each of the approaches described in the literature.
2 Cell Culture-based Methods
Viral isolation using cell culture based methods has been a gold standard to diagnose many viruses, especially to examine the infectivity and actual threat posed by a given contaminated sample (Hamza & Bibby, 2019; Hamza, Jurzik, Überla, & Wilhelm, 2011; Santos, 2015).
The main advantages in the use of cell culture are to amplify the amount of virus contributing for detection and identification, to produce infectious particles that can be further stored and characterized and to detect viral species that were even expected to be found at the beginning of the inoculation process (Santos, 2015). For the success of this protocol, the most important is the choice of the cell propagation system. When the virus is inoculated into a susceptible cell lineage, it can induce morphological modifications known as cytopathogenic effect (CPE) that are observed as a consequence of viral biosynthesis (Santos, 2015).
Viral isolation has some drawbacks during its execution that complicate its use as a routine. The work steps can be laborious, expensive; and some enteric pathogens such as hepatitis A virus (HAV), hepatitis E virus (HEV), and norovirus (NoV) are not easily cultivated (Hamza & Bibby, 2019; Hamza et al., 2011; Santos, 2015; Yang et al., 2011).
3 Immuno-based Methods
Direct and indirect immunofluorescence (IFD/IFI) and immunocytochemistry, also known as immunoperoxidase (IPX) staining, are useful techniques that confirm viral isolation in cell cultures when it is not possible to see a cytopathic effect (Bielanski & Dubuc, 1995). These techniques use antibodies to detect viral antigens and the reaction can be visualized through fluorescent stains like fluorescein in the case of IDF/IFI and carbazole for IPX. A fluorescence microscope is required; therefore, this need could be considered a disadvantage because the equipment is expensive so not all laboratory has one.
Many other innovative approaches are under development using antibodies for viral detection in environmental matrices. The viruses have antigenic properties that may be used for the production of specific antibodies; consequently, the viral antigen is recognized and attached by an antibody, forming an antigen-antibody complex (Rodríguez, Pepper, & Gerba, 2009). Following this approach, immunomagnetic separation (IMS) is a technique in which antibodies are bound to magnetic beads. The pathogen attaches to the antibody and this complex is removed from the sample with a magnet enabling its concentration (Hamza & Bibby, 2019; Rodríguez et al., 2009). This concentrate may be analyzed by different detection approaches, such as molecular methods and flow cytometer. Many studies have used IMS to improve viral detection in water samples (Grinde, Jonassen, & Ushijima, 1995; Haramoto, Kitajima, Katayama, & Ohgaki, 2010; Myrmel, Rimstad, & Wasteson, 2000; Yang et al., 2011). Another immune-based method, presented by Villamizar-Gallardo, Osma and Ortíz (2017), is a new technique for the direct fluoroimmunomagnetic detection, in which amino pink magnetic microparticles functionalized with monoclonal antibodies used to concentrate, separate and detect infectious Rotavirus (RV) in water samples. It seems to be a quick alternative, whereas even if performed along with the subsequent molecular characterization that is only for confirmation purposes, authors report that it takes no more than three hours. One disadvantage is the need for a confocal microscope.
With respect to immunochromatographic tests, there are few studies comparing the efficiency of commercial immunoassays for enteric viruses in stool samples, and the authors conclude that for clinical purposes, sensitivity is adequate, but for epidemiological studies, molecular methods are a better choice due to higher sensitivity (De Grazia et al., 2017; Kim et al., 2014; Rovida et al., 2013). These immunoassays were designed for detection in stool samples, and until this moment, no comparison was made to evaluate the efficacy in water samples. Despite the simplicity and inexpensiveness, these assays may not be sufficiently effective and sensitive for the use with environmental samples.
4 Conventional PCR
Nowadays, the molecular methods, which are based on protocols of nucleic acid amplification, have been used in most part of the studies that detect enteric viruses in water bodies (Girones et al., 2010; Hamza & Bibby, 2019; Haramoto et al., 2018). This advent started in the early 1990s with the standardization of the Polymerase Chain Reaction (PCR) technique that has become a specific, sensitivity and versatile method (Bonadonna et al., 2019; Hamza & Bibby, 2019). The molecular approaches have as the principal goal to achieve an increase in the accuracy and sensitivity of pathogens detection (Girones et al., 2010).
This technique became widespread in environmental analyses, especially because it gives the results in a short time, has high sensitivity and specificity, and the capacity to detect viruses that cannot be grown in cell culture (Haramoto et al., 2018), also, PCR can be used as a screening or characterization approach if coupled with sequencing (Bonadonna et al., 2019). However, PCR has some limitations, like the inability to quantify results, the co-concentration of PCR inhibitors, the ability to detect naked nucleic acids and the requirement to analyze PCR products in a gel electrophoresis that increase the analysis time (Bonadonna et al., 2019; Hamza et al., 2011).
5 Real-time PCR (qPCR)
Real-time PCR (qPCR), which is a quantitative version of conventional PCR, use specific primers and probes to achieve a significant knowledge about presence, quantity and distribution of pathogens in environmental samples with an important level of speed, sensitivity and specificity to detect viral genomes in low concentrations (Fong & Lipp, 2005; Girones et al., 2010; Hamza & Bibby, 2019). The protocol of qPCR could be easily standardized for the processing of many samples, thus allowing the analysis of a large number of samples in a single run. This characteristic support routine monitoring and risk assessment studies, since qPCR provides quantitative results, being an important tool to be used into risk assessment analysis (Girones et al., 2010).
Like other molecular methods, qPCR has also some drawbacks. This approach cannot discern between infectious and non-infectious viral particles and, because of that, the results may be overestimated in terms of individual risk of infection (Girones et al., 2010; Hamza & Bibby, 2019). Organic and inorganic contaminants are widely found in environmental samples; therefore, these inhibitors may affect the sensitivity of qPCR resulting in false negatives (Girones et al., 2010; Hamza & Bibby, 2019; Hamza et al., 2011). qPCR requires a standard curve with known quantification of the target gene to compare with unknown samples and, in consequence, this standard requires careful calibration and consistent source material to avoid incorrect results (Monteiro & Santos, 2017; Pinheiro et al., 2012).
Regarding to the viral infectivity issues, many studies have described different approaches that are combined and applied before qPCR to reduce or impede the possibility to detect damaged capsid consequently decreasing the detection of non-infectious viral particles. These techniques can be combined with cell culture as Integrated cell culture – PCR (ICC-PCR), immune-based method as IMS-qPCR, enzymatic treatments as nuclease and proteinase, and dye treatments as ethidium monoazide (EMA) and propidium monoazide (PMA) (Graiver, Saunders, Topliff, Kelling, & Bartelt-Hunt, 2010; Hamza & Bibby, 2019; Haramoto et al., 2010; Rigotto, Sincero, Simões, & Barardi, 2005).Even with these disadvantages, the benefits of this method have been contributing to spreading its use in environmental analysis. Therefore, qPCR is widely used in studies to investigate enteric viruses in different water sources like mineral water (Rodrigues dos Santos et al., 2015), river (Vecchia et al., 2015), stream (Girardi et al., 2019), wetlands (Gularte et al., 2017), seawater (Gularte et al., 2019; Staggemeier et al., 2017) and other kinds of environmental matrices (Gularte et al., 2019).
6 Digital PCR (dPCR)
Digital PCR (dPCR) is the new generation of molecular methods. dPCR works following a Poisson distribution therefore the target molecules are randomly dispensed into partitions and the PCR reactions are carried out (Bonadonna et al., 2019; Majumdar, Wessel, & Marks, 2015). As a result, when one or more target molecules are detected the partitions are positive and when no molecules are detected, the partitions are negative. Since positive partitions may contain more than one target molecule, Poisson statistics are applied to correctly estimate the number of the target within the analyzed sample presuming that each molecule has the same chance of landing in any partition (Majumdar et al., 2015).
The digital platform may consist of micro/nanofluidic-based or a droplet-based approach (Monteiro & Santos, 2017). It is noteworthy that dPCR over other nucleic acid detection based methods may allow the detection with no requirement of a known standard curve, and the quantification is more precise when compared to qPCR as well (Bonadonna et al., 2019; Hamza & Bibby, 2019). Another important advantage from dPCR over qPCR is the tolerance to inhibitors that are present in the samples (Hamza & Bibby, 2019).
As a challenge in the dPCR analyzes, there is a need to perform previous dilutions experiments in unknown samples to find the proper target detection range to achieve an acceptable level of precision. Therefore, it is important to use the original and diluted samples to ensure the extension of the supported dynamic range without loss of the target molecule (Majumdar et al., 2015).
The use of dPCR for analyzing environmental samples is not so widespread when compared with qPCR, but an increase in researches has been seen in the last years. Monteiro and Santos (2017) analyzed NoV into raw and treated wastewater samples by real-time reverse transcription PCR (RT-qPCR) and nanofluidic digital RT-PCR (RT-dPCR). Results were determined by both approaches without significant difference however data showed a lower variability in copy number of NoV for analysis made by RT-dPCR than RT-qPCR showing a higher precision in this new method. Sedji et al. (2018) carried out a research in a river located in Northeastern France using digital droplet PCR (ddPCR). In this study, Human mastadenovirus (HAdV) and NoV were positive in all river water samples (15/15). Steele, Blackwood, Griffith, Noble and Schiff (2018) evaluated water samples in two watersheds that discharge to surfing beaches in San Diego during storm water discharges using dPCR and RT-dPCR. NoV was detected in up 96% of samples, HAdV in 22% and enterovirus was not detected. Coudray-Meunier et al. (2015) carried out a study in lettuce and bottled water evaluating NoV and hepatitis A virus (HAV) by RT-dPCR and RT-qPCR. Viral recoveries were significantly higher by RT-dPCR than RT-qPCR for both viruses in the two samples. In addition, RT-dPCR proved to be more tolerant to inhibitory substances that were present in the lettuce samples.
7 Next-generation Sequencing (NGS)
Sanger, Nicklen and Coulson (1977) introduced the conventional DNA-sequencing approach. However, there are some limitations in this method, especially for environmental samples since these samples are complex and can include thousands of individuals from hundreds of species ranging from bacteria to higher eukaryotes. On the Sanger method is only possible to do the sequence of specimens individually, and in order to obtain more than one species, it is necessary to separate the individuals before the conventional sequencing (Hajibabaei, Shokralla, Zhou, Singer, & Baird, 2011). In addition, populations with low abundance are masked by the detection of the dominant population, preventing the knowledge of a largely unexplored diversity (Sogin et al., 2006). In contrast, nowadays the cost in NGS techniques have reduced, and consequently, this has accelerated the development of sequence-based metagenomics (Thomas, Gilbert, & Meyer, 2012).
Metagenomics consists in genetic analysis of the genomes contained in the environmental samples, and with different approaches, it is possible to compare many genomes from multiple taxa (Cantalupo et al., 2011; Thomas et al., 2012). With the advent in these analyses, it became possible to identify novel viral genotypes, species, genera and families. Additionally, since the virus’s genome is short, it is possible to obtain the near-complete or complete genome in a metagenomics analysis (Bibby et al., 2019; Ng et al., 2012). The next-generation sequencing (NGS) techniques facilitate the simultaneous analysis of millions of sequences (Fernandez-Cassi et al., 2018). Furthermore, NGS may be uses for the development of indicators and sentinels, new markers for microbial source tracking and observation of the microbially mediated processes (Tan et al., 2015).
The significant increase in human microbiome data and discovery of human-associated viruses by metagenomics have facilitated the development of new water quality monitoring tools with a focus on the viral indicators (Bibby et al., 2019). Raw sewage harbors a high diversity of viruses and has been already considered the most diverse viral biome by metagenomics analysis. In this study, they detected 234 known viruses, but most of the found genomes were considered novel viruses. Even with a wide variety identified, not all viruses present in the samples were detected because some viruses would be below the resolution of sequencing. In this way, it was possible to observe that no metagenomic method recovers and detects efficiently all types of virions, so, in some cases, the combination of approaches is required (Cantalupo et al., 2011).
Viruses do not have a conserved molecular marker across all species like bacteria. Given this to facilitate the viral metagenomes, the random-primer-based sequencing approaches have started to be applied in combination with NGS techniques. As carried out by Fernandez-Cassi et al. (2018), that to study the diversity of the genus Mastadenovirus in raw sewage, the samples were enriched using target broadly degenerated primers for the hexon region. This approach earned the detection of a wider range of AdVs with a higher variability of hosts.
In brief, with the metagenomic NGS, it is possible to discover new organisms and any portion of the genome can be detected. In addition, another approach called the targeted NGS can differentiate multiple species within one pathogen type. This new technology introduces many possibilities and it is just the beginning, so, it may still contribute a lot to the different environment analysis.
8 Biosensors
A device that converts a detection response into an electrical signal is called a sensor, thus, biosensors are a subgroup in which an analyte detection is carried out by a biological recognition element (Bahadır & Sezgintürk, 2017; Bonadonna et al., 2019). These devices have integrated receptor-transducer and the interaction between the biological element and target analyte generate a response (Bahadır & Sezgintürk, 2017). The biological recognition elements may be enzymes, antibodies, cells, aptamers, phages, single-stranded DNA, oligonucleotide probes or peptides (Bahadir & Sezgintürk, 2015; Bahadır & Sezgintürk, 2017; Bonadonna et al., 2019).
A biosensor is constituted by three elements that are a bioreceptor, a transducer and a signal-processing system (Bahadır & Sezgintürk, 2017). This approach may be classified based by their mode of physiochemical signal transduction; accordingly, the transducer is mostly characterized in electrochemical, optic, piezoelectric and thermometric transduction method (Bahadir & Sezgintürk, 2015; Bahadır & Sezgintürk, 2017; Bonadonna et al., 2019; Kumar, Hu, Singh, & Mizaikoff, 2018).
Biosensors have been implemented in different fields such as in the food industry, medical field, marine sector, etc. detecting contaminants like bacteria, viruses and pesticides, and biochemical parameters as glucose, lactate, lysine, and ethanol (Mehrotra, 2016). They have as major advantages a cheaper, fast and portable detection allowing a real-time and in situ contaminants monitoring (Bahadır & Sezgintürk, 2017), and they also must be highly specific and should be reusable providing better stability and sensitivity than traditional methods (Mehrotra, 2016).
The spread of pollutants and pathogens in the environment, especially in water sources, are increasing the need for the development of fast, cost-effective, sensitive and efficient analytical techniques (Bahadir & Sezgintürk, 2015; Kumar et al., 2018). Since traditional methods, as cell culture and molecular approaches, may be difficult; and expensive devices and specialized personnel are needed (Bahadir & Sezgintürk, 2015), biosensors have been developed to be a rapid, selective, portable and sensitive analytical approach for waterborne pathogens detection (Kumar et al., 2018).
The immuno-based method IMS, that was described above, has been already used in combination with a microfluidic technology for bacterial detection. This biosensor improved the capture efficiency because it enhanced the chances of interaction between antibody and antigen (Ahmed et al., 2016). The possibility to vary antibodies, beads types and sizes makes IMS a versatile and flexible technique (Ahmed, Rubahn, & Erdmann, 2016); therefore, the use of this combined technology to produce a lab-on-chip platform could be an important tool for the detection of enteric viruses in water samples as well.
Currently, some biosensors were already developed for the detection of viruses (Abadian, Yildirim, Gu, & Goluch, 2015; Janczuk-Richter et al., 2017; Jin et al., 2018). Even so, it is important to increase the researches in biosensors studies since many gaps still exist in water analyzes for enteric viruses that can be improved such as the presence of inhibitors and the need for prior concentration steps.
9 Conclusion
In this review, we analyze the pros and cons of the main methods used nowadays to detect viruses in environmental samples, especially in water samples. Traditional techniques, as cell culture and immuno-based methods, have largely been replaced by molecular approaches. However, they have still been used in the assessment of enteric viruses, especially because other techniques do not have the complete solution to analyze the viable viruses.
The use of molecular methods in the assessment of enteric viruses keeps improving due to the fact that these approaches have more advantages in the sensibility, specificity, and timer required to achieve results when compared with other techniques because of that these techniques are nowadays known as gold standard. The last advances in this field, such as qPCR and dPCR, have increased even more the attraction of the use for these tools, enhancing more in quantification and precision in the detection of viral particles in water samples.
NGS is the more current advance in the detection of viruses, being possible to detect more than one pathogen species with the same analysis, as well as to discover new species. Biosensors are also important for new devices that represent fast, specific, cheaper, and real-time approaches contributing to the advancement in the routine monitoring of water resources. Therefore, with the spread of those techniques, they will play a more important role in enteric viruses’ detection in a not far future.
The constant investment in researches that work for the development of new methods and improvement of the exited ones must improve continually. The field of environmental virology technologies still have some gaps that should be to work on for the next years in order to have accurate results, which can collaborate with the alternatives in investments in public policies aimed at the health of the population.
Acknowledgments
We thank the scholarships concession by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazilian Coordination for the Improvement of Higher Level Personnel (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (Foundation for Research Support of the State of Rio Grande do Sul (FAPERGS) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazilian National Council for Scientific Development (CNPq).
Conflict of Interest
The authors have no conflicts of interest.
Authors
Juliana Schons Gularte, PhD at Postgraduate Program in Environmental Quality at Feevale University. Member of Molecular Microbiology Lab (Feevale University) and One Health Lab (Feevale Techpark). The research group develops studies about contamination of water, soil, foods by enteric viruses and veterinary and human virology diagnoses.
Meriane Demoliner, PhD student at Postgraduate Program in Environmental Quality at Feevale University. Member of Molecular Microbiology Lab (Feevale University) and One Health Lab (Feevale Techpark). The research group develops studies about contamination of water, soil, foods by enteric viruses and veterinary and human virology diagnoses.
Ana Karolina Antunes Eisen, Master’s degree student at Academic Master’s degree in Virology at Feevale University. Member of Molecular Microbiology Lab (Feevale University) and One Health Lab (Feevale Techpark). The research group develops studies about contamination of water, soil, foods by enteric viruses and veterinary and human virology diagnoses.
Micheli Filippi, Biomedicine degree student at Feevale University. Member of Molecular Microbiology Lab (Feevale University) and One Health Lab (Feevale Techpark). The research group develops studies about contamination of water, soil, foods by enteric viruses and veterinary and human virology diagnoses.
Fernando Rosado Spilki, Full Professor at Feevale University, Brazil, Member of the Beyond Alliance for Knowlegde.
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