Investigating Virus Inactivation Mechanisms in Lagoon Systems. Rebecca Ives Michigan State University

Transcription

1 Investigating Virus Inactivation Mechanisms in Lagoon Systems Rebecca Ives Michigan State University

2 Presentation Outline Background Information on Virus Sampling and Processing Methods Results Summary & Conclusions Future Work

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4 Apollo 11, 20 July NASA

5 Apollo 14, 5 February NASA Apollo 11, 20 July NASA Transportation through a hostile environment Attachment to a surface

6 Apollo 11, 20 July NASA Apollo 14, 5 February NASA Rajagopala et al. 2011, BMC Microbiology 11: 213 Virus Image Credit: Graham Colm

7 Viruses are bio nanoparticles VIRUSES

8 Virus Obligate parasite cannot reproduce without a host cell Human Bacteria Other animal / insect Plant Multiple forms Nucleic acid: DNA, RNA Single strand, double strand Enveloped / Non-enveloped virus Other structures

9 Virus Obligate parasite cannot reproduce without a host cell Human Bacteria Other animal / insect Plant Multiple forms Nucleic acid: DNA, RNA Single strand, double strand Enveloped / Non-enveloped virus Other structures Potential indicator viruses Variable susceptibility to inactivation

10 Virus Obligate parasite cannot reproduce without a host cell Human Bacteria Other animal / insect Plant Multiple forms Nucleic acid: DNA, RNA Single strand, double strand Enveloped / Non-enveloped virus Other structures - Coliphage virus that infects E.coli Variable susceptibility to inactivation

11 Waterborne outbreaks, by year and etiologic agent United States, (N=609) Centers for Disease Control and Prevention. CDC Surveillance Summaries, November 19, MMWR 1993;42(No. SS-5).

12 Percentages of etiologic agents in outbreaks associated with drinking water (n = 780), 1971 to Craun et al. Clin Microbiol Rev. Jul 2010; 23(3):

13 On site Wastewater Systems in the Great Lakes IL IN MI MN NY OH PA WI Numbers of OWT ~1.7 million septic tanks in Michigan Gorman and Halvorsen,

14 Removal of virus via lagoon treatment EPA study of enteric virus removal Bausum et al While the present study provides a general verification of capacity of lagoon systems to remove enteric virus optimization of this virus removal capacity will require a better understanding of the mechanisms involved and their interactions

15 Factors influencing viral persistence Sobsey, M.D. and Meschke (2003) - WHO report Factor Temperature Light Target / Effect Protein denaturation, RNA damage, interference with enzymatic activity Radiation induced protein/nucleic acid breakage, formation of dimers, conformation changes, production of reactive oxidants Protective detergents Thermal stabilization of certain virus ph Capsid protein conformation, adsorption/elution from particles Microbial antagonism Predation, virocidal metabolites Ionic strength / salinity Chemical Organic material variable effects reported : some protective, some deleterious Capsid conformation change, opening of capsid Increased chlorine demand

16 Generalizations Viral inactivation is more rapid in microbially active environments than in sterile/pasteurized environments Viral inactivation is more rapid at higher temperatures Viral adsorption increases as ph decreases (Gerba, 1984) Viral adsorption increases as cation concentrations increase (Gerba, 1984) Adsorption of viral particles to organic particulates increases infectivity persistence (Gerba, 1975)

17 Cold (~0 C) Warm (15-30 C) Removal of microorganisms in aerated lagoons Locas et al Site 1 Site 2

18 Cold (~0 C) Warm (15-30 C) Removal of microorganisms in aerated lagoons Locas et al Somatic coliphage F + specific coliphage Human enteric virus

19 Lagoon Specific Literature on Virus Inactivation Mechanisms Ohgaki et al, 1986 Oxidation pond research Coliphage directly inactivated by sunlight only within 10cm of water surface Adsorption of coliphage to particulates (>0.45um) under aerobic conditions, desorption under anaerobic conditions Photosynthesis by algae during the day increases aerobic condition coliphage adsorb during the day and desorb at night Modeled data, duration of exposure=?

20 Research questions: What size fraction(s) are virus particles most associated with? Coliphage Full virus community Is coliphage inactivation/reduction by abiotic factors observable over small time intervals? Light Settling Heat

21 Coliphage vs Enteric Virus Assay Consideration Coliphage Enteric Virus Assay Cost ~$40 $600 - $1,000 Time to Result 24 hours 2-6 weeks Labor intensity Low High Technical expertise required Re-growth in environment possible? Low YES, if host is present High No

22 F + Specific Coliphage Attach to pilli on F + E.coli host Photo: Le revêtement cellulaire des cellules procaryotes

23 Coliphage Detection Double Agar Overlay (DAL) Majority of F+ phage detected in sewage by DAL are RNA phage

24 Viral Association with Particle Size: 3 discrete samples collected and composited from influent and each lagoon pond Sampling 6/26/2014

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28 Viral Association with Particle Size: Sampling and Processing Methods 0.2 µm filter ( +charge)

29 Viral Association with Particle Size: Sampling and Processing Methods 0.2 µm filter ( +charge)

30 F-specific Coliphage Sample Processing

31 Two sites Influent (A) Inactivation Study Anaerobic pond effluent (B) 3 discrete samples collected and composited

32 Why A and B? Inactivation Study High concentration of naturally occurring coliphage no need to amend samples Previous studies indicate the majority of inactivation occurs within the initial ponds

33 Inactivation: Sample Processing Sampling composite Dilution series Settling Shaking Settling Shaking Shaking 36 C

34 PFU/mL Virus association with particle size 200 F + specific Coliphage Concentration Unfiltered sample 180um filtrate 0.45um filtrate 0.20um filtrate 50 0 Influent Pond 1 Pond 2 Pond 3 Pond 4 Discharge

35 PFU/mL Virus association with particle size 200 F + specific Coliphage Concentration 3d Unfiltered sample 180um filtrate 0.45um filtrate 0.20um filtrate 50 0 Influent Pond 1 Pond 2 Pond 3 Pond 4 Discharge

36 Inactivation Data Results Anaerobic pond effluent (PFU/ml) Municipal wastewater influent (PFU/ml) Sampling Month Inactivation condition T 0 4 Hour 24 Hour 28 Hour T 0 4 Hour 24 Hour 28 Hour August w/o settling w/o light w/o settling with light with settling with light with settling w/o light September w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat * 289 October w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat November w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat

37 Inactivation Data Results Anaerobic pond effluent (PFU/ml) Municipal wastewater influent (PFU/ml) Sampling Month Inactivation condition T 0 4 Hour 24 Hour 28 Hour T 0 4 Hour 24 Hour 28 Hour August w/o settling w/o light w/o settling with light with settling with light with settling w/o light September w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat * 289 October w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat November w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat

38 Inactivation Data Results Anaerobic pond effluent (PFU/ml) Municipal wastewater influent (PFU/ml) Sampling Month Inactivation condition T 0 4 Hour 24 Hour 28 Hour T 0 4 Hour 24 Hour 28 Hour w/o settling w/o light w/o settling with light with settling with light August with settling w/o light September October w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat * 289 w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat w/o settling w/o light w/o settling with light with settling with light with settling w/o light w/o settling with heat November 1690

39 Example Data Set 1

40 Example Data Set 2

41 Inactivation Data Analysis SAS the mixed procedure Components Location (Influent, anaerobic pond effluent) Treatment 1 w/o settling w/o light 2 w/o settling with light 3 with settling with light 4 with settling w/o light 5 w/o settling with heat Time (4, 24, 28 hours)

42 Inactivation Data Analysis Type 3 Tests of Fixed Effects Effect Num DF Den DF F Value Pr > F Trt

43 Inactivation Data Analysis Least Squares Means Effect Trt Estimate Standard Error DF t Value Pr > t Trt Trt Trt Trt Trt

44 Inactivation Data Analysis Least Squares Means Effect Trt Estimate Standard Error DF t Value Pr > t Trt Trt Trt Trt Trt Treatment 1: w/o settling, w/o light

45 Inactivation Data Analysis Differences of Least Squares Means Effect Trt _Trt Estimate Standard Error DF t Value Pr > t Trt Trt Trt Trt Trt Trt Trt Trt Trt Trt

46 Conclusions Largest proportion of coliphage associated with particles in 0.45um - 180um size range None of the treatments examined resulted in statistically significant inactivation/reduction in F-specific coliphage

47 Future work Matthew Verbyla - Metagenomic analysis of virus communities associated with particle sizes >180um, um, um, and <0.2um Modifying inactivation rate study Inactivation in particle size fractions Increased duration of treatments

48 References Centers for Disease Control and Prevention. CDC Surveillance Summaries, November 19, MMWR 1993;42(No. SS-5). Craun GF, Brunkard JM, Yoder JS, et al. Causes of Outbreaks Associated with Drinking Water in the United States from 1971 to Clinical Microbiology Reviews 2010;23(3): doi: /cmr Gerba C.P. et al. Fate of Wastewater Bacteria and Viruses in Soil. Journal of the Irrigation and Drainage Division, Proceedings of the American Society or Civil Engineers. 101(IR3): (1975) Gerba C.P. & Bitton, G. Microbial Pollutants: Their Survival and Transport Pattern to Groundwater. In: Bitton, G., ed. Groundwater Pollution MicrobiologyNew York, NY, John Wiley & Sons, Inc., 1984, pp Locas A., Martinez V, and Payment P. Removal of human enteric viruses and indicator microoorganisms from domestic wastewater by aerated lagoons. Can. J. Microbiol 56: (2010) Sobsey M.D. and Meschke (2003) Virus Survival in the Environment with Special Attention to Survival in Sewage Droplets and Other Environmental Media of Fecal or Respiratory Origin. Report for the World Health Organization, 70 pages, Geneva, Switzerland.

49 References USEPA. Enteric Virus Removal in Wastewater Treatment Lagoon Systems. Project Summary Howard T. Bausum, Stephan A. Schaub, William E. Rose, and Paul H Gibbs. EPA 600 / S

50 Acknowledgements Bill Ribbens - City of Belding, Mi Matthew Verbyla - University of South Florida Timothy Stieve Michigan State University Rose Lab members

51 Questions?