Partnership Webcast Pepper Mild Mottle Virus: a Plant Pathogen With

Partnership Webcast

Pepper Mild Mottle Virus: A plant pathogen with a greater purpose in microbial water quality analyses!

Thursday, May 30, 2019 3pm - 4pm ET (1pm MT/2pm CT)

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Q&A at end of webinar

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3:00 – 3:05pm: Welcome, Purpose and Overview of WRF Partnership Webcast

3:05 – 3:10pm: Live Polling

3:10 – 3:40pm: Pepper Mild Mottle Virus: A plant pathogen with a greater purpose in microbial water quality analyses! Dr. Erin M. Symonds, University of South Florida

3:40 – 3:45pm: Progress and Activities on Viral Water Quality

Dr. Kyle Bibby, University of Notre Dame

3:45 – 4:00pm: Q & A

4:00 pm: Adjorn

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8 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Pepper Mild Mottle Virus: A plant pathogen with a greater purpose in microbial water quality analyses Dr. Erin M. Symonds Postdoctoral Researcher University of South Florida May 30, 2019

• Disease burden > 3 million ‘disability-adjusted life years’

• Economic burden > $USD 12 billion

Campylobacter Ascaris Giardia lamblia E.coli 0157:H7 Hepatitis A jejuni

Vibrio Cryptosporidium Norovirus Salmonella Rotavirus cholerae

Campylobacter Ascaris Giardia lamblia E.coli 0157:H7 Hepatitis A jejuni

Vibrio Cryptosporidium Norovirus Salmonella Rotavirus cholerae

Image: Getty Images/3DClinic Campylobacter Ascaris Giardia lamblia E.coli 0157:H7 Hepatitis A jejuni

Vibrio Cryptosporidium Norovirus Salmonella Rotavirus cholerae

Image: Getty Images/3DClinic Campylobacter Ascaris Giardia lamblia E.coli 0157:H7 Hepatitis A jejuni

Vibrio Cryptosporidium Norovirus Salmonella Rotavirus cholerae

Summarized in Lin & Ganesh 2013 Image: Canadian Food Inspection Agency2 14 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Indicators are used to study the fate of enteric pathogens in environmental waters & systems

• Have simple and affordable detection methods

• Have a decay and removal rate comparable to that of the pathogen of concern

• For use as a fecal pollution indicator, – not be present in environmental waters naturally – be present in polluted waters in concentrations correlated with the amount of fecal pollution

• Affordable & easy methods • Decay & removal rates similar to bacterial pathogens • As fecal pollution Fecal indicator bacteria (FIB) indicator, enterococci • Fecal coliforms correlated to illness at • Escherichia coli polluted beaches in temperate latitudes • Enterococci

• Can give results that are • false-negative because FIB are more susceptible to treatment • false-positive, in presence of secondary sources in the environment

• Cannot be used to identify the source of fecal pollution in the environment because FIB are excreted by all warm and some cold- blooded animals

Image: http://resizeandsave.online/dappy-May_2_2.html 19 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Viral indicators are used to study the fate of enteric viruses in the environment and in systems

Summarized in Lin & Ganesh 2013

Image: (left) Naranson3; (right) Wazzzup7up4 20 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Viral indicators are used to study the fate of enteric viruses in the environment and in systems

Coliphages are affordable indicators • F-specific RNA • Somatic

Summarized in Lin & Ganesh 2013

Adenovirus Norovirus

Polyomavirus Rotavirus

Summarized in Lin & Ganesh 2013 Images: RCSB PDB 6,5,8,7 22 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Sometimes reference pathogens are in lower concentrations, which make their detection difficult

• 102 – 104 per ml in domestic wastewater

Adenovirus • Consistently present in domestic wastewater

Polyomavirus

Lin & Ganesh 2013, Kitajima et al. 2014

• ≤ 103 per ml in domestic wastewater Norovirus • Variable concentrations in domestic wastewater Rotavirus

Kitajima et al. 2014, Lin & Ganesh 2013, Symonds et al. 2018

Adenovirus Norovirus

Polyomavirus Rotavirus Summarized in Lin & Ganesh 2013

Images: RCSB PDB 6,5,8,7, Wazzzup7up4 25 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Image: Aimme Blodgett, University of South Florida 26 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. What is the most abundant virus in human feces? - Dr. Mya Breitbart

Zhang et al. 2006

Colson et al. 2010, Zhang et al. 2006

Zhang et al. 2006, Summarized in Harwood et al. 2013

❑ First proposed as fecal pollution indicator by Rosario et al. 2009 • > 106 PMMoV per ml wastewater • 104 – 107 PMMoV per ml effluent

Rosario, Symonds, et al. 2009

❑ First proposed as fecal pollution indicator by Rosario et al. 2009 • > 106 PMMoV per ml wastewater • 104 – 107 PMMoV per ml effluent • Absent from surface waters without pollution • Detectable for 7 days post introduction to seawater

Rosario, Symonds, et al. 2009

Rosario, Symonds et al. 2009

Images: UF/IFAS9, USDA12, Sanchezn13, Vecteezy10, Wolfmann11, US EPA1 34 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Is PMMoV a useful indicator to study the fate of enteric viruses in environmental waters & in systems?

Verbyla, Symonds, et al. ES&T 2016 36 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Images: ME Verbyla 37 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Pathogen and indicator concentrations in Rocha River and riverbank filtration well

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well Well Well

Well

River River River

River

River River

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well Well Well

Well

River River River

River

River River

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well Well Well

Well

River River River

River

River River

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well Well Well

Well

River River River

River

River River

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well Well Well

Well

River River River

River

River River

2.0- to >3.5-log10 less abundant in the well water

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well

Well Well

Well

River

River River River

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well

Well Well

Well

River

River River River

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Below method

quantification limit

Concentration liter per Concentration

10 10

Log

Well

Well Well

Well

River

River River River

E.coli Coliphage Giardia Crypto Rotavirus Adenovirus Norovirus PMMoV HF183

Concentration liter per Concentration

10 10

Log

Well

Well Well

Well

River

River River River

❑ Consistently detected in high concentrations ❑ Geographic range • Americas • Europe • Asia • Africa • Australia

Summarized Symonds et al. 2018, Symonds et al. 2019, and Kitajima et al. 2018

it can be found in avian, dog, and cow feces.

Summarized Symonds et al. 2018, Symonds et al. 2019, and Kitajima et al. 2018

Images: UF/IFAS9, USDA12, Sanchezn13, Vecteezy10, Wolfmann11, US EPA1 50 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. PMMoV is a promising fecal pollution indicator in environmental waters Water Extent of Pooled Pooled Min Max type wastewater PMMoV pathogen PMMoV PMMoV pollution detection co-occurrence per liter per liter

groundwater point, 72% 23% 101 106 treated non-point 35% 5% 10-1 107 coastal point, 72% 61% 102 107 treated non-point 53% 12% 102 103 unknown 11% 0% 104 104 fresh point, 94% 72% 101 108 untreated point, 86% 68% 102 106 treated unknown 68% n/a 102 103

Summarized Symonds et al. 2018 51 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. PMMoV is a promising fecal pollution indicator in environmental waters Water Extent of Pooled Pooled Min Max type wastewater PMMoV pathogen PMMoV PMMoV pollution detection co-occurrence per liter per liter

Summarized Symonds et al. 2018 52 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. PMMoV is a promising fecal pollution indicator in environmental waters Water Extent of Pooled Pooled Min Max type wastewater PMMoV pathogen PMMoV PMMoV pollution detection co-occurrence per liter per liter

• Consistently higher concentrations make detection easier and overcomes methods limitations • Absent from unpolluted environmental waters • Presence in water reflects extent of wastewater pollution • Detection associated with illness above the US EPA health benchmark in recreational waters polluted with untreated wastewater

Summarized Symonds et al. 2018 and Kitajima et al. 2018 Image: US EPA1 54 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Is PMMoV a useful indicator to study the fate of enteric viruses in environmental waters & in systems?

3 Pond System

Alto Beni, Bolivia 3 Pond System

UASB Pond System UASB Pond System

Images: ME Verbyla 57 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. 3 Pond System UASB Pond System • Served 780 people • Served 1310 people • Theoretical hydraulic • Theoretical hydraulic retention retention time 26 days time 26-57 days

• 2-log10 – 6-log10 fecal • 2-log10 – 4-log10 fecal coliform coliform removal removal

Images: ME Verbyla 58 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. 3 Pond System UASB Pond System • Served 780 people • Served 1310 people • Theoretical hydraulic • Theoretical hydraulic retention retention time 26 days time 26-57 days

• 2-log – 6-log10 fecal coliform • 2-log – 4-log10 fecal coliform removal removal

Images: ME Verbyla 59 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Images: ME Verbyla 60 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Limited virus removal in the wastewater pond systems based on molecular methods

3 Pond System UASB Pond System

PMMoV concentrations were three orders of magnitude greater than those of norovirus and rotavirus throughout both treatment systems

3 Pond System UASB Pond System

• 3.1-log10 removal of • 0.8-log10 removal of culturable culturable viruses viruses

Virus-particle attachment may explain the difference… Large Mid

180 0.45 40 ml μm μm WW filter filter

Likely to Likely to Settle Shield

3 Pond System UASB Pond System

PMMoV, norovirus, & rotavirus were found in very low concentrations associated with particles likely to settle in both systems.

3 Pond3 Pond SystemSystem System UASB PondPond System System

• 3.1-log10 removal of • 0.8-log10 removal of culturable culturable viruses viruses • Less than 25% viruses • As many as 75% viruses associated with UV- associated with UV-shielding shielding particles in particles in UASB effluent facultative pond effluent

Image: ME Verbyla 65 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. PMMoV was a useful process indicator in our study on enteric virus removal in WTPs • PMMoV removal was similar to reference pathogens • PMMoV concentrations >>> than the reference pathogens; facilitating log-reduction calculations not possible the reference pathogens • High PMMoV concentrations facilitated identification of particle-associations and removal mechanisms

Image: Anthony Greco, University of South Florida 66 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. PMMoV is a promising process indicator of (waste)water treatment • Found in higher concentrations; thus, facilitates thorough (waste)water treatment efficiency assessments at all scales • PMMoV reduction is less than or equal to human enteric viruses of concern when measured by molecular methods

Summarized Symonds et al. 2018 Image: US EPA1 67 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. PMMoV is a promising indicator of enteric viruses An ideal indicator should… • Have simple and affordable detection methods

• Have a decay and removal rate comparable to that of the pathogen of concern

• For use as a fecal pollution indicator, – not be present in environmental waters naturally – be present in polluted waters in concentrations correlated with the amount of fecal pollution

• Have a decay and removal rate comparable to that of the pathogen of concern

• For use as a fecal pollution indicator, – not be present in environmental waters naturally – be present in polluted waters in concentrations correlated with the amount of fecal pollution

69 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Future research is still needed … • Expanding PMMoV geographic range • Investigating correlation to health risk and enteric viruses under different contexts • Overall method improvements – Distinguish infectious viruses – Simple, affordable, rapid, lab-free

Symonds & Breitbart CLEAN 2015

Bibby K, Crank K, Greaves J, Li X, Wu Z, Hamza IA, et al. Metagenomics and the development of viral water quality tools. NPJ Clean Water. 2019;2(1):9.

Kitajima M, Sassi HP, Torrey JR. Pepper mild mottle virus as a water quality indicator. NPJ Clean Water. 2018;1(1):19.

Symonds EM, Rosario K, Breitbart M. Pepper mild mottle virus: Agricultural menace turned effective tool for microbial water quality monitoring and assessing (waste)water treatment technologies. PLoS Pathog. 2019;15(4):e1007639.

Symonds EM, Nguyen KH, Harwood VJ, Breitbart M. Pepper mild mottle virus: A plant pathogen with a greater purpose in (waste)water treatment development and public health management. Water Res. 2018;144:1-12.

E.M. Symonds is supported by the US National Science Foundation under Grant OCE-1566562. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the US NSF.

Special thanks to all co-authors, in particular Dr. Mya Breitbart, Dr. Karyna Rosario, and Dr. Matthew Verbyla.

Thank you for joining us today

Colson P, Richet H, Desnues C, Balique F, Moal V, Grob J-J, et al. Pepper mild mottle virus, a plant virus associated with specific immune responses, fever, abdominal pains, and pruritus in humans. PLoS ONE. 2010;5(4):e10041. Harwood VJ, Staley C, Badgley BD, Borges K, Korajkic A. Microbial source tracking markers for detection of fecal contamination in environmental waters: Relationships to pathogens and human health outcomes. FEMS Microbiol Rev. 2013;38(1):1-40. Kitajima M, Sassi HP, Torrey JR. Pepper mild mottle virus as a water quality indicator. NPJ Clean Water. 2018;1(1):19. Kitajima M, Iker BC, Pepper IL, Gerba CP. Relative abundance and treatment reduction of viruses during wastewater treatment processes — Identification of potential viral indicators. Sci Total Environ. 2014;488–489:290-6. Lin J, Ganesh A. Water quality indicators: bacteria, coliphages, enteric viruses. Int J Environ Health Res. 2013;23(6):484-506. Ralston EP, Kite-Powell H, Beet A. An estimate of the cost of acute health effects from food-and water-borne marine pathogens and toxins in the USA. J Water Health. 2011;9(4):680-94. Rosario K, Symonds EM, Sinigalliano C, Stewart J, Breitbart M. Pepper mild mottle virus as an indicator of fecal pollution. Appl Environ Microbiol. 2009;75(22):7261-7. Shuval H. Estimating the global burden of thalassogenic diseases: Human infectious diseases caused by wastewater pollution of the marine environment. J Water Health. 2003;1:53-64. Symonds EM, Rosario K, Breitbart M. Pepper mild mottle virus: Agricultural menace turned effective tool for microbial water quality monitoring and assessing (waste)water treatment technologies. PLoS Pathog. 2019;15(4):e1007639. Symonds EM, Nguyen KH, Harwood VJ, Breitbart M. Pepper mild mottle virus: A plant pathogen with a greater purpose in (waste)water treatment development and public health management. Water Res. 2018;144:1-12. Symonds EM, Breitbart M. Affordable enteric virus detection techniques are needed to support changing paradigms in water quality management. CLEAN–Soil, Air, Water. 2015;43(1):8-12. Symonds EM, Verbyla ME, Lukasik JO, Kafle RC, Breitbart M, Mihelcic JR. A case study of enteric virus removal and insights into the associated risk of water reuse for two wastewater treatment pond systems in Bolivia. Water Res. 2014;65:257-70. Verbyla ME, Symonds EM, Kafle RC, Cairns MR, Iriarte M, Mercado A, et al. Managing microbial risks from indirect wastewater reuse for irrigation in urbanizing watersheds. Environ Sci Technol. 2016;50(13):6803-13. Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SWL, et al. RNA viral community in human feces: Prevalence of plant pathogenic viruses. PLoS Biol. 2006;4(1:108-18.

74 © 2019 The Water Research Foundation. ALL RIGHTS RESERVED. Media content sources 1. US EPA. https://www.epa.gov/eg/national-study-nutrient-removal-and-secondary-technologies 2. Canadian Food Inspection Agency (2011). Reproduced or adapted with the permission of the Canadian Food Inspection Agency, [2011]. 3. Naranson (2011). https://en.wikipedia.org/wiki/File:Ms2capsid_surface.png 4. Wazzzup7up (2009). https://commons.wikimedia.org/wiki/File:M._smegmatis_plaque.jpg 5. Image from the RCSB PDB (rcsb.org) of PDB ID:1HM; Prasad, B.V., Hardy, M.E., Dokland, T., Bella, J., Rossmann, M.G., Estes, M.K. Crystal structure analysis of Norwalk Virus capsid. DOI: 10.2210/pdb1IHM/pdb; H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242. 6. Image from the RCSB PDB (rcsb.org) of PDB ID:6CGV; Natchiar, S.K., Venkataraman, S., Nemerow, G.R., Reddy, V.S. National Institutes of Health/National Institute Of Allergy and Infectious Diseases. Revised crystal structure of human adenovirus. DOI: 10.2210/pdb6CGV/pdb; H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242. 7. Image from the RCSB PDB (rcsb.org) of PDB ID:3GZT; Chen, J.Z., Settembre, E.C., Harrison, S.C., Grigorieff, N.. VP7 recoated rotavirus DLP. DOI: 10.2210/pdb3GZT/pdb; H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242. 8. Image from the RCSB PDB (rcsb.org) of PDB ID:5FUA; Hurdiss, D.L., Morgan, E.L., Thompson, R.F., Prescott, E.L., Panou, M.M., Macdonald, A., Ranson, N.A.. Cryo-EM of BK polyomavirus. DOI: 10.2210/pdb5FUA/pdb; H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne. (2000) The Protein Data Bank Nucleic Acids Research, 28: 235-242. 9. UF/IFAS Pest Alert Web site/Pamela Roberts - Thriving Community of Pathogenic Plant Viruses Found in the Human Gut. PLoS Biology Vol. 4/1/2006, e15 https://dx.doi.org/10.1371/journal.pbio.0040015 10. Vecteezy. Attribution: Vector Design by Vecteezy! 11. Wolfmann (2018). https://commons.wikimedia.org/wiki/File:Toilet,_toilet_paper,_toilet_brush,_etc._in_a_bathroom_ in_small_hotel_in_Tysnes,_Hordaland,_Norway_2018-03-18_C._Also_wall_and_floor_tiles.jpg 12. USDA. https://www.usda.gov/media/blog/2011/11/17/poultry-classifications-get-21st-century-upgrade 13. Sanchezn (2008). https://commons.wikimedia.org/wiki/File:DefecatingSeagull2.jpg 14. Hph (2004). https://commons.wikimedia.org/wiki/File:Yungas04.jpg

• Sponsored by NSF award 1748019 • Webinars to communicate emerging viral water quality research results • Goal – move beyond E. coli as the target… • More to come annually!

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