Welcome to the October Edition of the 2020 M&R Seminar Series

Welcome to the October Edition of the 2020 M&R

Seminar Series

NOTES FOR SEMINAR ATTENDEES

• All attendees’ audio lines have been muted to minimize background

noise.

• A question and answer session will follow the presentation.

• Please use the Chat feature to ask a question via text.

• The presentation slides will be posted on the MWRD website after

the seminar.

Benito J. Mariñas, Ph.D.

Ivan Racheff Endowed Professor of Environmental Engineering

Dept of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign

Dr. Mariñas is Ivan Racheff Endowed Professor of Environmental Engineering, Department of

Civil and Environmental Engineering (CEE), University of Illinois at Urbana-Champaign (UIUC)

where he served as Department Head from 2014-2020. He is also Director of the Safe

Global Water Institute (2013-present) at UIUC and served as Director of the NSF Science

and Technology Center of Advanced Materials for the Purification of Water with Systems -

WaterCAMPWS (2012-14).

Dr. Mariñas’ research explores mechanistic aspects of chemical and ultraviolet light

disinfection processes, chemistry and toxicity of nitrogenous disinfection by-product (N-DBP),

with emphasis on N-DBP formation and control. He also does research on membrane

technologies for the control of water-borne pathogens and chemical contaminants, with

emphasis on the development of novel membrane materials.

Dr. Mariñas holds a B.S. degree in civil engineering from the Universidad Politecnica de

Madrid, Spain (1982); and M.S. (1985) and Ph.D. (1989) degrees in sanitary and

environmental engineering from the University of California at Berkeley. Before joining the

CEE faculty at UIUC in 1995, he was a faculty member (1989-1995) at the School of Civil

Engineering of Purdue University, West Lafayette, Indiana.

Dr. Mariñas was the recipient of the Arthur and Virginia Nauman Faculty Scholar award

(1998- 2005) at the Dept. of CEE of UIUC. He is a Fellow of the Association of

Environmental Engineering and Science Professors (2020), Member of the Academy of

Distinguished Alumni Academy, Dept of CEE, University of California at Berkeley (2019),

won Harold Munson Outstanding Teacher Award (1992), and Ross Judson Buck '07

Outstanding Counselor Award (1992) from the School of Civil Engineering at Purdue

University, and making the List of Teachers Ranked as Excellent by their Students multiple

times at the UIUC.

SAFE GLOBAL WATER INSTITUTESAFE GLOBAL WATER INSTITUTE

Advances in Kinetics, Mechanisms, and

Detection of Viral Pathogens

Toward Developing Smart Water

Disinfection Systems

Benito J. MariñasDirector, Safe Global Water Institute (SGWI)

Ivan Racheff Professor, Department of Civil & Environmental Engineering

SAFE GLOBAL WATER INSTITUTE

SAFE GLOBAL WATER INSTITUTESAFE GLOBAL WATER INSTITUTESAFE GLOBAL WATER INSTITUTE

Sensor development

Real time detection

of infectious viruses

Gall et al., PLoS Pathog

2015, 11 (6), e1004867

Inter/Transdisciplinary Research Approach

Inactivation kinetics

1.0 E-5

1.0 E-4

1.0 E-3

1.0 E-2

1.0 E-1

1.0 E+0

0 20 40 60 80 100 120 140 160 180

Surv

ival

Rat

io (

N/N

0)

UV Dose (mJ/cm2)

214 nm220 nm228 nm232 nm239 nm248 nm254 nm260 nm270 nm280 nm

HAdV-2

UV224

UV254

Binding

DNA

Replication

Inhibited

UV280

E1A mRNA

Transcription

Reduced

E1A mRNA

Transcription

Inhibited

Binding

DNA

Replication

Inhibited

Untreated

Virus

Binding

DNA

ReplicationE1A mRNA

Transcription

or

NH2Cl?

?

?

Kwanrawee Sirikanchana, Martin Page, Aimee Gall, Bernardo Vazquez, Dana Al-Qadi, Kelley Gonçalves,

Shiliang Tian, Wen Cong, Ana S. Peinetti, Anisa Hardin

Joanna L. Shisler, Yi Lu

pH 7

CT (mg min/L)

0.0 2.5 5.0 7.5

N/N

0

10-5

10-4

10-3

10-2

10-1

100

pH 8

CT (mg min/L)

0 5 10 15

pH 9

CT (mg min/L)

0 20 40 60

5oC

15oC

25oC

USEPA SWTR 5oC

15oC

25oC

HOCl

UV

Protein/Genome transformation

Replication cycle disruption


Pathogens: Human Adenovirus 2

(non-enveloped capsid

~90 nm; dsDNA ~36 kbp)

Coxsackievirus B5

(enterovirus;non-enveloped

capsid ~30 nm;

+ssRNA ~7.5 kb)

Disinfectants: Free chlorine

Monochloramine

Experiments performed

with synthetic buffered

solutions in batch

reactors

Viability assessment by

plaque assay using

human lung A549

carcinoma cells (HAdV-

2), or Buffalo Green

Monkey Kidney

(BGMK) cells (CoxV-

B5)

TEM image of human

adenovirus type 2

Broad Objective: Have better control of

viruses in drinking

water Determination of

inactivation kinetics

Inactivation mechanisms

Sensitive methods to

detect infectious viruses

(ultimately including those

for which cell culture is

not available, like human

norovirus)

Viral Pathogens, Disinfectants & Objectives

Martin Page, Kwanrawee Sirikanchana, Aimee Gall, Bernardo Vazquez, Kelley Gonçalves, Wen Cong, Anisa Hardin

Joanna L. Shisler


INACTIVATION KINETICS


Inactivation of Human Adenovirus 2

with Free Chlorine

pH 8.1

CT (mg min/L)

0.0 0.1 0.2 0.3

pH 9.2

CT (mg min/L)

0.0 0.5 1.0 1.5

pH 7.4

CT (mg min/L)

0.0 0.1 0.2

N/N

0

10-5

10-4

10-3

10-2

10-1

100

1oC

14oC

30oC

USEPA SWTR 1oC

14oC

30oC

M.A. Page, J.L. Shisler, B.J. Marinas, Wat. Res., 2009, 43, 2916-2926


Inactivation of Coxsackievirus B5

with Free Chlorine

pH 7

CT (mg min/L)

0.0 2.5 5.0 7.5

N/N

0

10-5

10-4

10-3

10-2

10-1

100

pH 8

CT (mg min/L)

0 5 10 15

pH 9

CT (mg min/L)

0 20 40 60

5oC

15oC

25oC

USEPA SWTR 5oC

15oC

25oC

A. Hardin, W. Cong, B.J. Marinas, in preparation



with Free Chlorine

pH 8.1

CT (mg min/L)

0.0 0.1 0.2 0.3

pH 9.2

CT (mg min/L)

0.0 0.5 1.0 1.5

pH 7.4

CT (mg min/L)

0.0 0.1 0.2

N/N

0

10-5

10-4

10-3

10-2

10-1

100

1oC

14oC

30oC

USEPA SWTR 1oC

14oC

30oC

M.A. Page, J.L. Shisler, B.J. Marinas, Wat. Res., 2009, 43, 2916-2926



with Monochloramine

A.M. Gall, J.L. Shisler, B.J. Marinas, Environ. Sci. Technol. Lett., 2016, 3, 185-189

CT (mg×min/L)

0 5000 10000 15000

N/N

0

10-5

10-4

10-3

10-2

10-1

100

CT (mg×min/L)

0 2000 4000 6000

CT (mg×min/L)

0 250 500 750 1000

N/N

0

10-5

10-4

10-3

10-2

10-1

100

5°C 15°C 30°C

pH 9

pH 7pH 6

pH 8 pH 9 pH 8pH 7pH 6

VirusesUSEPA SWTR

pH 6pH 7pH 8pH 9


INACTIVATION MECHANISMS:

LIFE CYCLE STEP INHIBITION


HAdV-2 and CoxV-B5 Replication Cycles

HAdV-2

Selected HAdV-2 (90 nm, ~36,000 bp) Replication cycle events:

Attachment (fiber to CAR, penton base to integrin?) – 0 h p.i.

Internalization with endosome

Virion uncoating and endosome rupture

DNA release into nucleus

Early mRNA (E1A) synthesis – 1-2 h p.i.

DNA replication and late mRNA (hexon) synthesis – 5-8 h p.i.

Cell lysis – 48 h p.i. DNA: Quantitative Polymerase Chain Reaction (qPCR)

mRNA: Reverse Transcriptase qPCR (RT-qPCR)

HAdV-2 genome showing selected amplicons

CoxV-B5

Selected CoxV-B5 (30 nm, ~7,500 b) replication cycle events:

Attachment (VP2&3 to DAF VP1, VP3, VP4 to CAR?) – 0 h p.i.


Virion uncoating

RNA release from endosome

RNA synthesis 4-5 h p.i

Viral lytic and non-lytic (vesicle) release – 6 h p.i.

CoxV-B5 genome showing selected amplicons


Comparison of Free Chlorine


with relative quantity of early (E1A)

and late (hexon) DNA and RNA

• Experiments performed at pH 7,

15oC

• Adenovirus inactivation at levels

up to 99.99% did not result in loss

of amplicon integrity or ability to

attach

• However, synthesis of viral DNA

and early E1A and late hexon

gene transcription were inhibited

CT (mgCl2×min/L)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

N/N

0,

E1

A/E

1A

0,

or

Hex

on

/Hex

on

0

10-6

10-5

10-4

10-3

10-2

10-1

100

E1A/E1A0 at 4 h p.i. (Hexon-not detected)

E1A/E1A0,

E1A/E1A0,

E1A/E1A0,

Hexon/Hexon0 at 12 h p.i.



N/N0

N/N

0,

E1

A/E

1A

0,

or

Hex

on

/Hex

on

0

10-6

10-5

10-4

10-3

10-2

10-1

100

E1A/E1A0,

E1A/E1A0,

E1A/E1A0,

E1A/E1A0,

E1A/E1A0,






N/N0

N/N

0,

E1

A/E

1A

0,

or

Hex

on

/Hex

on

0

10-6

10-5

10-4

10-3

10-2

10-1

100

N/N0

E1A/E1A0

Hexon/Hexon0

DNA Amplicon Integrity

DNA Replication

RNA Synthesis

A.M. Gall, J.L. Shisler, B.J. Marinas,

Environ. Sci. Technol., 2015, 49, 4584-4590


Comparison of Monochloramine


with relative quantity of early (E1A)

and late (hexon) DNA and RNA

• Experiments performed at pH 8,

15oC

• Adenovirus inactivation at levels

up to 99.99% did not result in loss

of amplicon integrity or ability to

attach

• However, synthesis of viral DNA

and early E1A and late hexon

gene transcription were inhibited

CT (mgCl2×min/L)

0 500 1000 1500 2000

N/N

0,

E1

A/E

1A

0,

or

Hex

on

/Hex

on

0

10-6

10-5

10-4

10-3

10-2

10-1

100

N/N

0,

E1

A/E

1A

0,

or

Hex

on

/Hex

on

0

10-6

10-5

10-4

10-3

10-2

10-1

100

N/N

0,

E1

A/E

1A

0,

or

Hex

on

/Hex

on

0

10-6

10-5

10-4

10-3

10-2

10-1

100

E1A/E1A0

Hexon/Hexon0

N/N0

E1A/E1A0, Hexon/Hexon

0 at 4 h p.i.


0 at 12 h p.i.


0 at 24 h p.i.


0 at 36 h p.i.

N/N0


0 at 0 h p.i.

E1A/E1A0 at 4 h p.i. (Hexon-not detected)

E1A/E1A0,

E1A/E1A0,

E1A/E1A0,




N/N0

DNA Amplicon Integrity

DNA Replication

RNA Synthesis

A.M. Gall, J.L. Shisler, B.J. Marinas, Environ. Sci. Technol.

Lett., 2016, 3, 185-189


Comparison of Free Chlorine Inactivation of Coxsackievirus

B5 with relative quantity of RNA

• Experiments performed at pH 7, 15oC

• In contrast to the observation for

adenovirus, there is measurable loss of

amplicon integrity and ability to attach at

higher Coxsackievirus B5 inactivation

levels

• Similar to the observation for synthesis

of DNA genes for adenovirus, synthesis

of viral RNA genes for Coxsackievirus

B5 were inhibited

A. Hardin, W. Cong, B.J. Marinas, in preparation

2C - 106 b - translate into protein 2C which rearranges the

cellular membranes for the purpose of viral replication.

3D -149 b - translate into protein 3D, which is an RNA-

dependent RNA polymerase

LR (long range) - 1235 b located near the end of the genome

CT (mgCl2×min/L)

0 1 2 3 4 5

N/N

0, 2

C/2

C0, o

r 3D

/3D

0

10-5

10-4

10-3

10-2

10-1

100

N/N0

2C/2C0, 3D/3D

0 at 0 h p.i.

2C/2C0, 3D/3D

0 at 4 h p.i.

2C/2C0, 3D/3D

0 at 6 h p.i.

2C/2C0, 3D/3D

0 at 8 h p.i.

2C/2C0, 3D/3D

0 at 2 h p.i.

RNA Replication

N/N

0, 3

D/3

D0, o

r L

R/L

R0

10-5

10-4

10-3

10-2

10-1

100

N/N0

3D/3D0

LR/LR0

RNA Amplicon Integrity


HAdV-2 and CoxV-B5 Replication Cycles

HAdV-2

Selected HAdV-2 (90 nm, ~36,000 bp) Replication cycle events:

Attachment (fiber to CAR, penton base to integrin) – 0 h p.i.


Virion uncoating and endosome rupture

DNA release into nucleus

Early mRNA (E1A) synthesis – 1-2 h p.i.

DNA replication and late mRNA (hexon) synthesis – 5-8 h p.i.

Cell lysis – 48 h p.i. DNA: Quantitative Polymerase Chain Reaction (qPCR)

mRNA: Reverse Transcriptase qPCR (RT-qPCR)

HAdV-2 genome showing selected amplicons

CoxV-B5

Selected CoxV-B5 (30 nm, ~7,500 b) replication cycle events:

Attachment (VP2,VP3 to DAF, VP1, VP3, VP4 to CAR) – 0 h p.i.


Virion uncoating

RNA release from endosome

RNA synthesis 4-5 h p.i

Viral lytic and non-lytic (vesicle) release – 6 h p.i.

CoxV-B5 genome showing selected amplicons

?

?

??

? ?


INACTIVATION MECHANISMS:

CAPSID PROTEIN TRANSFORMATION


Modifications of Human Adenovirus 2

Capsid Proteins with Free Chlorine

(Reddy and Nemerow, 2014)

• Proteins targeted were: fiber,

penton base, hexon

• The genes of the adenovirus fiber,

penton base, and hexon were

expressed in gene-modified E. coli

• 1 uM of each protein monomer

(with 571, 582, 967 residues) was

reacted with 10, 100, 1,000 uM

free chlorine for 5 min (assuming

no chlorine decay CT=3.6, 36, 360

mg min/L) before quenching with

excess sodium thiosulfate

• Proteins digested (Trypsin) and

residues analyzed by LC-MS/MS

S. Tian, A.M. Gall, W. Cong, J.L. Shisler,

B. Marinas, Y. Lu, in preparation


0 100 200 300 400 500 600 700 800 900

10100

1000

N V1 V1DE1 V1 V2 V2 V2 VCFG1 FG2

DE

2

VC

Cl 2

:Pro

tein

Mola

r R

atio

Amino Acid

B

A

Cl2:Protein

Molar Ratio10 100 1000

Graphical depictions of the HAdV-2 fiber, penton based, and

hexon (bottom) fiber (expressed in E. coli) modifications by

free chlorine exposure

Ribbon diagrams showing modified

residues (0.5-5%) in red (unmodified

residues shown in green; peptides not

detected shown in tan; 2-12%) with

chlorine exposure: Top: fiber tail, shaft, and head

domains: CAR D1 binding &

flexible shaft regions at mid and

high CT;

Middle: penton base regions

involved in pentamer formation,

RGD loop, RGD integrin binding

regions at lowest CT;

Bottom: hexon N terminus, viral

jellyrolls, extended loops, and the

viral jellyroll connector at all CTs.

S. Tian, A.M. Gall, W. Cong, J.L. Shisler,

B. Marinas, Y. Lu, in preparation

Met≈Cys>Trp Met≈Cys>His>Trp>Tyr>Asn≈Gln


INFECTIOUS VIRUS DETECTION:

APTAMER-BASED SOLID STATE

NANOPORE SENSOR DEVELOPMENT


In vitro selection of an aptamer toward infectious HAdV-2

Target preparation

Human Adenovirus 2

DNA library

1014 sequences

45 random nucleotides

11 Rounds

A.S. Peinetti, W. Cong, A. Gall, B.J. Marinas, O. Azzaroni, Y. Lu, submitted

Free Chlorine

pH 8.15, 22oC

CT≈2.3 mg min/L


Solid State Nanopore Sensor

• The aptamer showed great selectivity for the infectious (active) HAdV

• The nanopore (900±100 nm → 55±5 nm) amplified the signal several orders of magnitude

G. Perez-Mitta, A.S. Peinetti, M.L. Cortez, M.E. Toimil-Molares, C. Trautmann, Omar Azzaroni, NanoLett. 2018, 18, 3303-3310A.S. Peinetti, W. Cong, A. Gall, O. Azzaroni, B.J. Marinas, Y. Lu, submitted

• 2 hr equilibration

with viruses

• electrochemical

cell: 0.1 M KCl,

±1V


Solid State Nanopore Sensor

G. Perez-Mitta, A.S. Peinetti, M.L. Cortez, M.E. Toimil-Molares, C. Trautmann, Omar Azzaroni, NanoLett. 2018, 18, 3303-3310A.S. Peinetti, W. Cong, A. Gall, O. Azzaroni, B.J. Marinas, Y. Lu, submitted

• Comparison to inactivation data by plaque assay

• Preliminary Selectivity Tests

• No background interference (same results with buffer, tap water, WW effluent)

Method; concentration

(mean ± SD, n=3, pfu/ml)

Sample

#

%

inactivationNanopore system Plaque-assay RPD (%)

1 90 528 ± 613 ±

2 99 101 ± 26 94 ±

3 99.9 12 ± 2 9.2 ±


Sensor development

Real time detection

of infectious viruses

Gall et al., PLoS Pathog

2015, 11 (6), e1004867

Inter/Transdisciplinary Research Approach

Inactivation kinetics

1.0 E-5

1.0 E-4

1.0 E-3

1.0 E-2

1.0 E-1

1.0 E+0

0 20 40 60 80 100 120 140 160 180

Surv

ival

Rat

io (

N/N

0)

UV Dose (mJ/cm2)

214 nm220 nm228 nm232 nm239 nm248 nm254 nm260 nm270 nm280 nm

HAdV-2

UV224

UV254

Binding

DNA

Replication

Inhibited

UV280

E1A mRNA

Transcription

Reduced

E1A mRNA

Transcription

Inhibited

Binding

DNA

Replication

Inhibited

Untreated

Virus

Binding

DNA

ReplicationE1A mRNA

Transcription

or

NH2Cl?

?

?

Kwanrawee Sirikanchana, Martin Page, Aimee Gall, Bernardo Vazquez, Dana Al-Qadi, Kelley Gonçalves,

Shiliang Tian, Wen Cong, Ana S. Peinetti, Anisa Hardin

Joanna L. Shisler, Yi Lu

pH 7

CT (mg min/L)

0.0 2.5 5.0 7.5

N/N

0

10-5

10-4

10-3

10-2

10-1

100

pH 8

CT (mg min/L)

0 5 10 15

pH 9

CT (mg min/L)

0 20 40 60

5oC

15oC

25oC

USEPA SWTR 5oC

15oC

25oC

HOCl

UV

Protein/Genome transformation

Replication cycle disruption


Benito J. Mariñas

[email protected]

cee.illinois.edu

THANK YOU!

Documents

Welcome to the October Edition of the 2020 M&R Seminar Series