ENABLING ADAPTIVE UV AND SOLAR-BASED DISINFECTION

SYSTEMS TO REDUCE THE PERSISTENCE OF VIRAL PATHOGENS IN WASTEWATER FOR SUSTAINABLE

REUSE

Thanh H. (Helen) Nguyen (thn@Illinois.edu), Jeremy Guest, Joanna ShislerUniversity of Illinois

1

RD83582201-0

Transport and Survival of Pathogens in Aquatic Environment: Water Reuse Application

http://www.water.ca.gov/recycling/2

Rotavirus: One of the Most Common Diarrhea Causing Viruses

Rotavirus, Pesavento et al., 2001)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC29265/figure/F2/

3

Kiulia et al., Pathogens. 2015 May 13;4(2):229-55.

Occurrence and Emission of Rotavirus to Surface Waters

Rotavirus Serotypes

4Desselberger, et al.(2009) Rotavirus and rotavirus vaccines. British Med Bull (2009) 90 (1)

• The capsid consists of VP2, VP6, and VP7• VP6: 7 serological groups (A to G)• VP7 (glycoprotein): 14 G serotypes• VP4 (hemagglutinin protein): P serotypes

Human and Animal Rotavirus Found in Lettuces from AZ

5Aw, et al., International Journal of Food Microbiology, 223, 16 April 2016

Reuse of Treated Wastewater

6

Urbana-Champaign Wastewater Treatment Facility

Google earth image

SEW

Cortland Reclamation for Irrigation

Google earth image

Pond 2

Project Objectives1. Determine the molecular mechanisms

responsible for virus inactivation 2. Determine factors required for effective virus

inactivation by natural sunlight and UVC3. Develop pond and UVC design guidelines to

achieve reliable virus inactivation and elucidate trade-offs across and within dimensions of sustainability.

7

Sunlight Is The Most Important Factor

Causing Disinfection In Surface Water Sunlight

2006; Davies-Colley et al. 1997, 1999; Da Silva, A.K, et al., 2008;

http://www.thewatertreatments.com/waste-water-treatment-filtration-purify-sepration-sewage

1. Direct UVB Damage Causes direct effects on DNA by inducing dimerization of pyrimidine bases, leading to the formation of photo-products that may block DNA replication

280 300 320 340 360 380 4000

20

40

60

80

100

UVAUVB

SunlightSolar Simulator

Irrad

ianc

e (W

/m2 /n

m)

Wavelength (nm)

8

http://www.thewatertreatments.com/waste-water-treatment-filtration-purify-sepration-sewage

Indirect UVA Damage:2. Exogenous

Manuel A. et al., 2007.

280 300 320 340 360 380 4000

20

40

60

80

100

UVAUVB

SunlightSolar Simulator

Irrad

ianc

e (W

/m2 /n

m)

Wavelength (nm)

9

*CO32-H2O2

OH

DOM 1DOM*

3DOM*O2

1O2 O2

-

http://www.thewatertreatments.com/waste-water-treatment-filtration-purify-sepration-sewage

Indirect photo-oxidation damage is caused by generating ROS that damage the genome, proteins, and/or lipids. ROS formation is catalyzed by endogenous sensitizers.

Indirect UVA Damage:2. Exogenous3. Endogenous

280 300 320 340 360 380 4000

20

40

60

80

100

UVAUVB

SunlightSolar Simulator

Irrad

ianc

e (W

/m2 /n

m)

Wavelength (nm)

10

, Pesavento et al., 2001)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC29265/figure/F2/

Xenon arc lamp with filters

Experimental Setup

300 315 330 345 3600.0

0.1

0.2

0.3

0.4

0.5

UIUC Solar Simulator 8/15/2012 at 12 pm (Sunny & Clear) 3/8/2013 at 12 pm (Sunny & Clear) 3/3/2013 at 12 pm (Cloudy)

Irrad

ianc

e (W

/m2 /n

m)

Wavelength (nm)

11

Hypotheses1. The phenotypic and genotypic differences

between virus strains can be used to identify the molecular mechanisms responsible for viral susceptibility to solar and UVC inactivation

2. a) Photo-oxidation of viruses depends on the reactions between viruses and reactive radicals formed upon irradiation of wastewater, b) direct effect of UVC irradiation on viruses depends on the irradiation wavelength and the specific UV absorbance (SUVA) of the wastewater

3. Human health risk and life cycle ecological impacts can simultaneously be reduced by leveraging solar disinfection. 12

Insignificant Endogenous Inactivation of Human and Porcine Rotavirus at 25°C

0 1 2 3

-1.0

-0.5

0.0

sensitizer-free solution

Porcine Rotavirus

Human Rotavirus

25°C

Log

Inac

tivat

ion

(FFU

/mL)

Irradiation Time (hr)

Romero-Maraccini, et al. ES&T 2013 13

Human Rotavirus Is More Susceptible to Exogenous Inactivation by Direct Sunlight UVA than Porcine

Rotavirus at 25°C

0 1 2 3

-1.0

-0.5

0.020 mg C/L

20 mg C/L

Porcine Rotavirus

Human Rotavirus

25°C

Log

Inac

tivat

ion

(FFU

/mL)

Irradiation Time (hr)

Romero-Maraccini, et al. ES&T 2013 14

Indirect UV-A inactivation of Porcine Rotavirus is important at 40-50ºC

10 20 30 40 50 60

0.0

0.5

1.0

1.5

2.0

0 mg C/L

k ob

s(hr-1

)

Temperature (°C)

20 mg C/L

dark control

Romero, et al. ES&T 2011 15

Inactivation of Human Rotavirus at 25°C Correlates with Inactivation of Porcine Rotavirus 50°C

0.0 0.5 1.0 1.5 2.0 2.50.0

0.5

1.0

1.5

Sensitizer-free Buffer Control Blavet River DOM (20 mg C/L) HA Suwannee River DOM (20 mg C/L) EfOM - hydrophilic fraction (20 mg C/L) EfOM - hydrophobic fraction (20 mg C/L) NaNO2 (43 µM) NaNO3 (0.59 mM)

y= 0.61x+ 0.06

R2=0.98

p < 0.0001

Hum

an R

otav

irus

k obs (

h-1)

Porcine Rotavirus kobs (h-1)

Romero-Maraccini, et al. ES&T 2013 16

Reactive Oxygen Species in Natural Water

17

O2

1O2 singlet oxygen

DOM 1DOM*

3DOM*triplet excited

O2-

H2O2hydrogen peroxide

*CO32-

OHHydroxyl radical

NOx-

OHHydroxyl radical

Triplet DOM Can Inactivate Human Rotavirus

0 10 20 30

0.2

0.4

0.6

0.8

1.0R2= 0.99p = 0.004

Hum

an R

otav

irus

k obs (

h-1)

3'-MAP (mg/L)

3-MethoxyacetophenoneTriplet DOM* sentisizer

18

Singlet Oxygen 1O2 Does Not Inactivate Human Rotaviruses

Blavet SR95 HA XAD80.0

0.5

1.0

1.5L-Histidine: 1O2 quencher

Human Rotavirus Porcine Rotavirus

Rat

ios

of R

otav

irus

k obs_

quen

cher/k

obs v

alue

s

DOM solutions

0 10 20 300.0

0.5

1.0

1.5

sensitizer-free buffer control; Blavet River DOM (20 mg C/L); HA SR95 DOM (20 mg C/L); XAD4 EfDOM (20 mg C/L); XAD8 EfDOM (20 mg C/L)

p = 0.3

Hum

an R

otav

irus

k obs (

hr-1)

1O2 Concentration (x10-14 M)

19

Can H2O2 Inactivate Rotavirus?

• 0, 6, and 60 µM of H2O2 did not show significant inactivation of rotavirus at 50°C in the dark.

• The measured [H2O2] at 50°C in 20 mg/L TOC was ~ 6 µM after the 3 hrs of the experiment.

• Thus, H2O2 is not directly responsible for rotavirus inactivation at 50°C.

20

Need to look at OH radicals

21

O2

1O2 singlet oxygen

DOM 1DOM*

3DOM*triplet excited

O2-

H2O2hydrogen peroxide

*CO32-

OHHydroxyl radical

NOx-

OHHydroxyl radical

Hydroxyl Radical Is Responsible for Inactivation of Human Rotavirus Wa:

Hypothesis 2a Was Proven

pH=8.0 and 25ºC

0.00 0.01 0.02 0.03 0.04 0.050.0

0.5

1.0

1.5

Blavet River DOM

Suwannee River DOM

Hydrophobic fraction EfDOM

NO2-

NO3-

hydrophilicfraction EfDOMControl

R2 = 0.96p < 0.0001

Hum

an R

otav

irus

Inac

tivat

ion

Rat

e C

onst

ant (

h-1)

Phenol first-order decay constant (h-1)

Romero-Maraccini, et al. ES&T 201322

Indirect UVA Inactivation of Human Rotavirus In Waste Stabilization Pond Water

pH=8.0 and 25ºC

0.00 0.01 0.020

1

2

3

R2 = 0.90p = 0.0007

Buffer Control; Pond 1; Pond 2 Pond 3; Poind 4; Pond 5; Pond 6

Hum

an R

otav

irus

k obs (

h-1)

Phenol kexp (h-1)

http://erikaquiroz.com/map-of-arizona/

23

Inactivation Rate Constants Correlated with SUVA

0 1 2 3 4 50.0

0.5

1.0

1.5

R2 = 0.80p = 0.025

R2= 0.86

p = 0.016

Filled symbols: Porcine Rotavirus; Empty symbols: Human Rotavirus)

/ Sensitizer-free Buffer Control/ Hydrophobic EfOM/ Hydrophilic EfOM/ Suwannee River NOM/ Blavet River NOM

Specific UV254nm Absorbance

Rota

virus

kob

s (h-1

)

0.0 0.5 1.0 1.5

0

1

2

3

Rot

aviru

s k ob

s (h-1

)

SUVA

Buffer Control; Pond 1; Pond 2 Pond 3; Pond 4; Pond 5; Pond 6

y ( )

24

Porcine Rotavirus Is More Resistant Than Human Rotavirus

0 1 2 3

-1.0

-0.5

0.020 mg C/L

20 mg C/L

Porcine Rotavirus

Human Rotavirus

25°C

Log

Inac

tivat

ion

(FFU

/mL)

Irradiation Time (hr)

20 30 40 50 600369

12

50

60 Dark Porcine Rotavirus (OSU) Human Rotavirus (Wa)

Temperature (°C)Ro

tavir

us k

obs (

hr-1)

Romero-Maraccini, et al. ES&T 2013 25

Protein % identity of OSU to Wa

VP1 97VP2 97VP3 94

VP4 * 69VP5 75VP8 54VP6 93VP7 79

Significant Differences in Structural Protein Sequences of Human RV Wa and Porcine RV OSU

VP4

VP7VP6

VP2

VP1/VP3

dsRNA segment

Manuel A. et al., 2007.

Romero-Maraccini, et al. ES&T 2013 26

Rotavirus Reassortment

Modified after Barry C Buckland, Nature Medicine 11, S16 - S19 (2005)

Human Rotavirus Animal Rotavirus

Human-animalreassortant

27

Aw, et al., International Journal of Food Microbiology, 223, 16 April 2016

John-T-Patton, Rotavirus diversity and evolution in the post vaccine world, Discovery Medicine, 2012

Distribution of RV Genotypes Reported to the WHO Surveillance Network in 2010

28

Studied Rotavirus Serotypes

29Desselberger, et al.(2009) Rotavirus and rotavirus vaccines British Med Bull (2009) 90 (1)

RV Glycoprotein Strains HostG1 Wa HumanG2 S2 HumanG3 YO HumanG4 ST3 HumanG5 OSU Porcine

• VP6 : 7 serological groups (A to G)

• VP7 (glycoprotein) :14 G serotypes

Effects of Solar Irradiation and Temperature Treatments on the Life Cycle

of Human Rotavirus Wa

0.0 0.5 1.0

-2

-1

0

57°C

Full spectrum + NOM

Full spectrum

UVA-vis+ NOM

UVA-vis

Dark Control

Hum

an R

otav

irus

Log

Inac

tivat

ion

(FFU

/mL)

Irradiation Time (h)

280 300 320 340 360 380 4000

20

40

60

80

100

UVAUVB

SunlightSolar Simulator

Irrad

ianc

e (W

/m2 /n

m)

Wavelength (nm)

Romero-Maraccini, et al. AEM, 201530

Schematic of the rotaviruslife cycle

Stepsinvestigated

ch

Genomeintegrity

• Targeted a portion of theNSP3 segment

Binding ability

• Tracked bound rotavirus to host cell receptors by quantifyingthe NSP3 gene by RT-PCR

RNA synthesis

• Assessed RNA synthesis by quantifying the levels of NSP3 gene copies generated inside the host by RT-PCR

Cell lysis• Conducted the

infectivity assay

Rotavirus Life Cycle

Host cell

RNA synthesis

Binding Entry

Uncoating of VP7 and VP4 proteins

Transcription (+RNAs)

Protein translation

Core Assembly

Genome replication(dsRNA)

Particlematuration

Cell lysis

Host cell

Rotavirus

Romero-Maraccini, et al. AEM, 2015 31

Effects of Treatments on RV Life Cycle

57 degrees C (dark)

Full spectrum+SRNOM

Full spectrum

UVA-vis + SRNOM

UVA-vis

0.0 0.5 1.0

Relative contribution to the overall inactivation

Direct genome damage Binding* RNA synthesis* Post RNA synthesis

Romero-Maraccini, et al. AEM 2015 32

Year 1 Summary1. Hydroxyl radical produced by organic matter

triggers rotavirus Wa inactivation (hypothesis 2a is proven for one strain).

2. Genetically different strains of rotavirus have different susceptibility toward solar disinfection.

3. When using solar disinfection, the decrease in RNA synthesis was responsible for approximately one-half of the decrease in infectivity, suggesting that other mechanisms, including posttranslational, contribute inactivation.

33

Year 2 and 3 Plan1. Determine the molecular mechanisms

responsible for inactivation of different RV strains;

2. Determine factors required for effective virus inactivation by UVC; and

3. Develop pond and UVC design guidelines to achieve reliable virus inactivation and elucidate trade-offs across and within dimensions of sustainability.

34

Publication Supported by EPA Project RD83582201-0

• Romero-Maraccini, OC, Shisler, JL, Nguyen., TH., Solar and Temperature Treatments Affect the Ability of Human Rotavirus WaTo Bind to Host Cells and Synthesize Viral RNA, Applied and environmental microbiology, 81 (12), 4090-4097, 2015, doi:10.1128/AEM.00027-15.

• Feng, Z., Lu, R., Yuan, B., Zhou, Z., Wu, Q., Nguyen, T.H., Influence of solution chemistry on the inactivation of particle-associated viruses by UV irradiation, Colloids and Surfaces B: Biointerfaces, 148, pp. 622–628, 2016, doi: j.colsurfb.2016.09.025.

• Fuzawa, M., Ku, K.-M., Palma-Salgado, S. P., Nagasaka, K., Feng, H., Juvik, J. A., Sano, D., Shisler, J.L., and Nguyen, T. H., Effect of Leaf Surface Chemical Properties on the Efficacy of Sanitizer for Rotavirus Inactivation, Applied and Environmental Microbiology, 82 (20), pp 6214-6222, 2016, doi: 10.1128/AEM.01778-16

35