Metagenomic Approaches for Detecting Viral Diversity in Water Environments
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Metagenomic Approaches for Detecting Viral Diversity in Water Environments Camille McCall, M.ASCE1; and Irene Xagoraraki, Ph.D., M.ASCE2 Abstract: Methods for detecting and monitoring known and emerging viral pathogens in the environment are imperative for understanding risk and establishing regulatory standards in environmental and public health sectors. Next-generation sequencing (NGS) has uncovered the diversity of entire microbial populations, enabled discovery of novel organisms, and allowed pathogen surveillance. Metagenomics, the sequencing and analysis of all genetic material in a sample, is a detection method that circumvents the need for cell culturing and prior understanding of microbial assemblies, which are necessary in traditional detection methods. Advancements in NGS technologies have led to subsequent advancements in data analysis methodologies and practices to increase specificity, and accuracy of metagenomic studies. This paper highlights applications of metagenomics in viral pathogen detection, discusses suggested best practices for detecting the diversity of viruses in environmental systems (specifically water environments), and addresses the limitations of virus detection using NGS methods. Information presented in this paper will assist researchers in selecting an appropriate metagenomics approach for obtaining a comprehensive view of viruses in water systems. DOI: 10.1061/(ASCE)EE.1943-7870.0001548. © 2019 American Society of Civil Engineers. Introduction monitoring the presence of viral pathogens in environmental sam- ples (Bibby and Peccia 2013; Ramírez-Castillo et al. 2015). The increase in viral-related diseases, lack of medications to In the early 1950s, Dulbecco and Vogt (1954) demonstrated that treat viral infections, low infectious dose, and high mortality rates it was possible to perform plaque assays on human viruses as with among children, elderly, and the immunocompromised is of global bacteriophages, viruses that infect bacteria. Briefly, viruses of vary- concern. Sewage-treatment plants serve as a significant exposure ing dilutions are applied to a susceptible cell monolayer. A semi- pathway for viral pathogens. Viral pathogens are known to resist solid media (e.g., agar) is then added to the monolayer to increase treatment and are released into receiving water bodies, posing a viscosity. This restricts the spread of the virus to only neighboring major threat to human health (Xagoraraki et al. 2014; Wigginton cells within the monolayer. Infected cells lyse and form holes et al. 2015). Therefore, detection and surveillance of viruses in (i.e., plaques) in the monolayer, which can be seen with the naked water environments is of growing importance. Conventional virus eye or by staining to increase visibility (Taylor 2014). Each plaque detection methods include indirect methods involving indicator is known as a plaque-forming unit (PFU) and can be counted and organisms, traditional cell-culture techniques like plaque assays expressed as a concentration. Furthermore, observing CPEs in in- and viral-induced cytopathic effects (CPEs), and molecular tech- fected cells is a widely used culture-based method for virus identi- niques such as polymerase chain reaction (PCR). This paper briefly fication in clinical diagnostics. Changes in cell morphology on a cell summarizes traditional methods for virus detection and addresses monolayer are observed microscopically, and the viral agent is iden- modern techniques, namely, next-generation sequencing, in detail. tified based on its known CPE. CPEs can appear in various forms, Indirect detection methods have been established for sug- including cell shrinking, swelling, and cell clustering (Leland and gesting the presence of pathogens in environmental systems. In par- Ginocchio 2007). ticular, measuring the concentration of indicator organisms such Traditional culture-based methods allow for the isolation of as coliforms and coliphages is a standard water quality practice purified virus stock and are effective at determining infectivity for monitoring the presence of enteric pathogens. The survivability and concentration. However, these methods are limited by long of indicator organisms depends on a number of environmental incubation periods and the inability to culture most viruses. PCR factors, and they are unable to determine the identity of a specific has become a standard molecular method for detecting and quan- pathogen. Although the concentration of these organisms are suit- tifying viruses in environmental media. This method bypasses the able for suggesting the occurrence of fecal contamination, they do need for cell culturing, provides rapid detection, and is more effi- not always correlate with the presence or absence of viral patho- cient for analyzing environmental samples (Xagoraraki et al. 2014). gens. Direct detection methods are required for classifying and Downloaded from ascelibrary.org by Michigan State University on 06/04/19. Copyright ASCE. For personal use only; all rights reserved. Quantitative PCR (qPCR) enables DNA to be simultaneously quan- tified and the concentration calculated given a standard curve. 1Ph.D. Student, Dept. of Civil and Environmental Engineering, Additionally, sequence-specific probes can be used to increase the Michigan State Univ., East Lansing, MI 48824. ORCID: https://orcid.org specificity of DNA amplification. Quantitative reverse transcription /0000-0001-7516-8391. Email: [email protected] (RT-qPCR) involves a collective set of tools to amplify and quantify 2 Associate Professor, Dept. of Civil and Environmental Engineering, ribonucleic acid (RNA) viruses such as influenza, rotavirus, and Michigan State Univ., East Lansing, MI 48824 (corresponding author). others. In RT-qPCR, RNA is converted into complementary deoxy- Email: [email protected] Note. This manuscript was submitted on July 14, 2018; approved on ribonucleic acid (DNA) (cDNA) using an enzyme, reverse tran- December 7, 2018; published online on May 22, 2019. Discussion period scriptase. The cDNA can then be used as a template for qPCR. open until October 22, 2019; separate discussions must be submitted Despite the advances of molecular methods, many of these for individual papers. This paper is part of the Journal of Environmental techniques rely on well-known primers and a priori knowledge Engineering, © ASCE, ISSN 0733-9372. of the sample’s microbiome. This limits the ability to establish a © ASCE 04019039-1 J. Environ. Eng. J. Environ. Eng., 2019, 145(8): 04019039 comprehensive view of viral populations or the discovery of novel during an outbreak in western Uganda in 2007. The Bundibugyo pathogens in water and other environmental systems (Fancello et al. ebolavirus was found to be 35%–45% divergent from previously 2012). The need for direct human pathogen detection and viral di- characterized Ebola virus genomes and was therefore difficult to versity assessments in various ecosystems has led to the develop- detect using molecular methods. NGS assisted in the identification ment of a new generation of technologies. of this new Ebola virus species with a fatality rate of approximately Next-generation sequencing (NGS), or high-throughput sequenc- 36% within 10 days of RNA extraction. The new genome was ing (HTS), circumvents the limitations of traditional methods by us- constructed from the identified novel sequence, and molecular ing advanced technologies to sequence genes or genomes of entire techniques were used to confirm the presence of Bundibugyo organisms or microbial communities without the need for culturing ebolavirus in isolates of infected patients, thus facilitating rapid iso- or prior knowledge of the microbial structure of a sample. Several lation and control of the disease (Towner et al. 2008). DNA sequencing methods were introduced in the 1970s (Wu and Moreover, metagenomics has provided a comprehensive look Taylor 1971; Sanger et al. 1973; Maxam and Gilbert 1977). Building into viral diversity in environmental samples and has identified on previous methods, in 1977, Frederick Sanger developed a method novel and newly classified viral pathogens in several water and for DNA sequencing using chain-terminating inhibitors (Sanger et al. water-related habitats. Bibby and Peccia (2013) found high occur- 1977). Limitations of the earlier Sanger method included sequencing rences of herpesvirus and papillomavirus, along with newly discov- only up to 1,000 base pairs (bps) of DNA, or short reads (Heather ered picornaviruses, in Class B sewage sludge samples using and Chain 2016). In order to combat this limitation, larger DNA metagenomic analysis. Parechovirus and coronavirus were also de- sequences were sheared into smaller fragments, which were then tected in biosolids (Bibby et al. 2011), which highlights the risk of sequenced individually and reassembled through computational biosolids as a means for pathogen transport due to their use in land methods, a process known as shotgun sequencing (Anderson 1981; applications. Metagenomic analysis also identified human viral Gardner et al. 1981). Improvements and variations of the Sanger pathogens rhinovirus, enterovirus, parechovirus, and aichi virus shotgun method have since been introduced into the NGS methods in reclaimed water discharged from public sprinklers, fountains, widely used today, thus establishing Sanger as one of the founding and spigots at a plant nursery (Rosario et al. 2009). However, these pioneers of genomic sequencing. viruses obtained a low percent identity to reference