HUMAN VIRUS IN FECALLY CONTAMINATED WATER, THE VIROME OF URBAN SEWAGE
Fernandez-Cassi X1, Rusiñol M1, Timoneda N1,Aguado D1, Gonzales-Gustavson E1, Bofill-Mas S1, Abril JF3, Girones R1.
1Laboratory of Virus Contaminants of Water and Food, Microbiology Department, Biology Faculty, Barcelona University, Catalonia, Spain.
2Computational Genomics Lab (Compgen), Genetics Department & Biomedicine Institute (IBUB), Biology faculty, Barcelona University, Catalonia, Spain.
Many viral infectious diseases are transmitted by consumption or contact with water or food contaminated with the discharge of untreated or even treated sewage. Water quality, and therefore its uses, is evaluated according to FIB levels (Fecal Indicator Bacteria). It is widely accepted that these indicators do not always correlate with enteric viruses. Water which accomplishes the criteria stablished for FIB can be used after being treated for different purposes, such as irrigation of fresh produce, representing a threat for consumers as it may contain some viral pathogens such us noroviruses among others. The use of molecular techniques can give us a clue when searching for a concrete pathogen but might therefore miss some others.
The introduction of new techniques such as metagenomics in the analysis of water and food can give us important information of all the viruses present in a sample at the same time.
- To improve the procedure for analyzing environmental samples by metagenomics approaches: Next Generation Sequencing (NGS) of a DNA library prepared from environmental samples is the final step of a procedure in which viruses present in environmental samples are concentrated and their genomes previously amplified. Several approaches have been conducted in order to obtain representatives libraries presenting low levels of interfering genomes and inhibitors. This study has also been focused on the optimization of the bioinformatics analysis pipeline used for analysis and identification of the obtained sequences.
- To improve our knowledge on the excreted virome and to identify the viruses that may represent a risk for the population associated to the consumption of fecally contaminated water or food.
Viral particles from 10L raw sewage water samples collected from a wastewater treatment plant in the area of Barcelona were concentrated using a skimmed milk flocculation based procedure.
In order to eliminate all free RNA and/or DNA present in the samples, RNAse and/or DNAse treatments were evaluated by using qPCR. RNAse treatments were also applied to compare the effectiveness of constructing total nucleic acids (NA) libraries and DNA libraries separately.
Also, NA amplification protocols and different amounts of DNA were tested in order to evaluate sensitivity of the assay.
The DNA obtained was used for constructing a randomized library with Nextera kit and DNA fragments analyzed with Illumina Miseq platform.
The genomic reads obtained were cleaned and filtered by quality and sequence complexity. The resulting sequences were afterwards blasted against viral reference genome database in order to determine known viruses present on the samples, as well as to define novel taxonomical units. In a parallel process under development, raw reads were assembled into putative contigs to reduce sequence redundancy and to facilitate the posterior mapping to the genomes of known species.
The evaluation of the RNAse treatment by qPCR showed little effect therefore only DNAse treatment was applied to eliminate free nucleic acids.
The analysis of DNA vs. analysis of total nucleic acids did not improve the sensitivity of the assay for detection of DNA viruses.
More reads were also observed when using total nucleic acids instead of only DNA.
The study of raw sewage samples collected in different seasons in the area of Barcelona allowed the identification of more than 25 different viral families. The majority of reads mapped to ssRNA viruses belonging to Virgaviridae family. This family is known to infect plants and some species like tomato mosaic virus (TMV) and pepper mild mosaic virus (PPMoV) are widely known to be present in high numbers in human feces.
Some viral families such as Adenoviridae or Polyomaviridae, prevalently present in raw sewage, seemed to be scarcely detected using metagenomics approaches. This suggests the existence of bias towards some viral families due to a higher concentration of other families or biases due to the molecular techniques used during library preparation. This last hypothesis seems more than reasonable as many reads were mapping the same viral region probably due to the low initial nucleic acid concentrations and the need to amplify using PCR to construct the library.
Among the 25 families identified, 9 comprise human viral pathogens. The most commonly found viruses in urban sewage such us adenoviruses, astroviruses, enteroviruses, rotaviruses, as well as noroviruses were detected in raw sewage samples using a metagenomics approach.
The use of this technique allowed the detection of the recently described virus Salivirus/klassevirus, a new genus belonging to Picornaviridae family in urban sewage. Other members of the picornaviridae family such as the emerging enterovirus EV-71, which can cause food and mouth disease (HFMD), and aichi virus which has been considered to be an etiological agent of gastroenteritis have been identified.
The use of metagenomics allowed the description of several astroviruses including the new emerging variants MBL2 and HMO which usually are not screened by conventional diagnostic tests.
The human viral fecal markers Human adenovirus (HAdV) and the JC polyomavirus (JCPyV) could only be found in one of the samples analyzed. Finally, sequences belonging to Circoviridae and Parvoviridae families were also found showing a wide prevalence of these viruses in raw sewage,
Using treated sewage for applications, such as crops irrigation, must be further analyzed considering the risk of transmission of human viruses and also the role of urban sewage in the transmission of infectious pathogenic plant viruses.
Metagenomics is a useful tool to screen the viral diversity present in raw sewage water samples: it can detect known viruses that may represent a risk for the population through the consumption of contaminated water or food; and it can identify potential new viruses present in these samples too. Our protocol was able to find virus causing gastroenteritis belonging to the families Caliciviridae, Astroviridae, Picornaviridae, Adenoviridae, and Reoviridae. Moreover, virus belonging to the families Papillomaviridae, Polyomaviridae,Circoviridae, and Parvoviridae, whose fecal-oral transmission is unclear and, in some cases, also their pathology were also found.