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Screening for Potential Viral Pathogens in Wastewater Effluent

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Screening for Potential Viral Pathogens in Wastewater Effluent Screening for potential viral pathogens in wastewater effluent and activated sludge using metagenomics analysis Evan O’Brien, Mariya Munir, Terence Marsh, and Irene Xagoraraki Abstract Results Sampling Location Despite recent rapid advancements in water and wastewater treatment technologies, S2 S4 S7 S9 S11 S14 waterborne pathogens still remain as one of the major environmental threats to human Taxonomy EL_AD EL_BD EL_AS TC_AD TC_BD TC_AS Numerous potential human pathogenic viruses health. Monitoring of all pathogens with conventional methods is not feasible due to Viruses 29706 29479 28763 32316 30182 27878 (Poxviridae, Herpesvirales, Adenoviridae, dsDNA viruses, no RNA stage 28633 28728 27813 31349 29189 27042 time and cost constraints. In this study, viral diversity of two wastewater treatment plant Caudovirales 18179 23026 16122 24253 23565 17832 Polyomaviridae, Coronaviridae) found in samples effluents, a conventional activated sludge (CAS) facility and a membrane bioreactor Phycodnaviridae 3643 1429 4512 1929 1394 3077 Mimiviridae 3048 814 3420 1068 1131 1911 Large proportion of sequence reads unaffiliated with (MBR) facility, are investigated using metagenomics. Diversity analysis does not unclassified dsDNA viruses 1358 1197 1557 1521 774 2196 existing genomic data unclassified dsDNA phages 821 1716 570 1878 1665 1087 provide quantitative data on pathogen loads or infectivity but it provides a list of Poxviridae 465 158 508 250 149 409 Greatest portion of affiliated sequences associated with potentially pathogenic viruses that need to be considered in more detail. The most Iridoviridae 208 129 179 79 148 102 Ascoviridae 247 45 238 94 42 169 bacteriophages; very small proportion affect vertebrates abundant potential human viral pathogen observed in our study belongs to taxonomic Baculoviridae 167 59 215 99 52 103 Marseilleviridae 239 0 253 0 102 0 order Herpesvirales. Other potentially pathogenic viruses detected in this study include Herpesvirales 118 62 95 45 66 73 Poxviridae, Adenoviridae, and Coronaviridae. Metagenomic analyses in this study also Asfarviridae 18 39 19 64 28 17 Bicaudaviridae 36 14 34 14 13 24 revealed that a large proportion of sequences could not be assigned to taxonomic Nudiviridae 35 0 46 0 19 0 Tectiviridae 12 10 10 27 11 18 affiliations even at the phylum/class levels and thus are most likely to be derived from Lipothrixviridae 21 7 23 11 7 10 Top-left: Sampling device used in experiment. Bottom-left: Illimuna novel, uncharacterized microbes. This study provides guidance on which viral Ligamenvirales 23 0 28 0 11 0 Nimaviridae 4 9 2 4 9 5 HiSeq DNA Sequencer. Right: Map of sampling locations. pathogens to monitor in the effluents of WWTPs especially in case of wastewater reuse. Rudiviridae 2 3 5 4 4 2 Polydnaviridae 6 2 1 3 2 3 EAST LANSING WWTP TRAVERSE CITY WWTP It also indicates the need to standardize metagenomics analysis methods in terms of Plasmaviridae 2 1 3 3 1 4 Wastewater treatment Conventional Activated Sludge Membrane Biological Reactor sample preparation and data analysis. Adenoviridae 2 5 0 1 4 0 process (Biological treatment) (CAS) (MBR) Corticoviridae 2 3 0 2 3 0 Sludge Retention Time (SRT) 14 days 7.58 days Polyomaviridae 0 0 1 0 0 0 ssDNA viruses 163 43 109 149 43 98 Capacity 18.8 MGD* 17.0 MGD Inoviridae 153 34 101 72 34 85 Average flow 13.4 MGD 8.5 MGD Parvoviridae 0 0 0 47 0 6 Discharge Rate 14.1 MGD 4.0 MGD unclassified ssDNA viruses 2 3 5 16 4 6 Disinfection Chlorine (Cl) Ultra-Violet (UV) Methods Microviridae 8 5 3 9 5 1 Circoviridae 0 1 0 4 0 0 Sample Collection: Effluent before (BD) Sequencing: Virus-enriched DNA isolated Geminiviridae 0 0 0 1 0 0 Table 2: Wastewater treatment plant characteristics. Retro-transcribing viruses 81 25 69 27 21 29 Figure 1: Metagenome summary (from MetaVir) and after disinfection (AD) and activated from the samples was sequenced on an Retroviridae 62 16 68 15 13 29 Caulimoviridae 19 9 1 12 8 0 sludge samples (AS) were collected from Illumina HiSeq platform. Sequences were ssRNA viruses 21 9 16 10 9 24 ssRNA positive-strand viruses, East Lansing (EL) WWTP and Traverse assembled into contigs and diversity 21 9 16 9 9 24 no DNA stage City (TC) WWTP in 2013. Argonite analysis was completed using the MetaVir Potyviridae 14 7 14 7 7 15 Conclusions Closteroviridae 1 0 1 1 0 2 (electropositive cartridge) filters were used analysis platform. The alignment modules Nidovirales 6 2 1 1 2 7 in the filtration and concentration of the Bowtie2 and SAMTools were then used to Mesoniviridae 5 2 0 1 2 2 Coronaviridae 1 0 1 0 0 5 Most abundant potential human samples for viral isolation. Approximately offer further confirmation of the presence ssRNA negative-strand viruses 0 0 0 1 0 0 dsRNA viruses 4 2 1 2 2 0 pathogens found in samples belong to 400 liters of effluent samples were passed of viruses of interest. Cystoviridae 2 2 1 2 2 0 through the sampler at a rate of about 11- Endornaviridae 1 0 0 0 0 0 taxonomic order Herpesvirales; other unclassified dsRNA viruses 1 0 0 0 0 0 Activated 12 L/min. Activated sludge (AS) samples Effluent Water Sample unclassified phages 658 655 577 763 827 661 potentially pathogenic human viruses were collected in two 1 L Nalgene bottles, Sludge Sample unclassified viruses 125 1 153 0 76 0 Filtration unassigned viruses 14 6 16 5 6 9 detected include Adenoviridae, unclassified virophages 2 7 2 10 7 13 mixed together in the laboratory and Argonite filters unclassified archaeal viruses 5 3 7 1 2 2 Poxviridae, and Coronaviridae. treated for virus isolation from each There is need for standardization of WWTP. Elution and Concentration Table 1: Taxonomic comparison heat map based on contigs best Figure 2: Breakdown of affiliated sequences by virus host (from MetaVir) methods for sample preparation in Sample Processing: All effluent and Viral DNA Extraction BLAST hit numbers (from MetaVir). metagenomic analysis to allow sludge samples collected were eluted 12– comparison among different studies Illumina Sequencing (HiSeq) 24 hours after initial sampling. Further The significant proportion of concentration of the solution was done by Sequence Reads Quality Control: Trimming References unaffiliated sequences means that more Trimmomatic placing 500 mL into a bottle and investigation is required to improve the centrifuged for 15 min at 2500×g at 4 °C. • ASTM. 2002. Standard practice for recovery of viruses from wastewater sludges, ASTM D4994-89. ASTM, West Conshohocken, PA. DeNovo Assembly The supernatant was loaded into a 60 mL IDBA-UD • Kuo, D. H. W., Simmons, F. J., Blair, S., Hart, E., Rose, J. B., & Xagoraraki, I. (2010). Assessment of human adenovirus removal in a full-scale membrane bioreactor robustness of available genomic data syringe and passed through a 0.22 μm treating municipal wastewater. Water research, 44(5), 1520-1530. Metagenomics still only a screening Annotation and Taxonomic Classification • Roux, S., Tournayre, J., Mahul, A., Debroas, D., & Enault, F. (2014). Metavir 2: new tools for viral metagenome comparison and assembled virome analysis. BMC sterilized filter for removal of bacteria, MetaVir2 bioinformatics, 15(1), 76. tool; further analysis is necessary to fungi and other contaminating agents. • USEPA, 2001. Manual of Methods for Virology (Chapter 14). EPA 600/4–84/013 Office of Water, U.S. Environmental Protection Agency, Washington, DC. determine infectivity of pathogens in Virus DNA was extracted using a MagNA Genome Alignment Evaluation Bowtie2, SAMTools Acknowledgments wastewater Pure Compact DNA extractor. We would like to thank the managers of the East Lansing and Traverse City Wastewater Treatment Plants for providing samples and information needed for this study. A very special thanks to the Bioinformatics Center for Education and Productivity (BiCEP) at the Institute for Cyber-Enabled Research (ICER) at Michigan State University for the bioinformatics support and assistance provided. .
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