WATERBORNE VIRAL PATHOGENS AND METHODS OF DETECTION IN
WASTEWATER: A MOLECULAR MAGAZINE
By
ABIGAIL GRACE ALBRIGHT____________________A Thesis Submitted to The W.A. Franke Honors College
In Partial Fulfillment of the Bachelor’s degree
With Honors in
Molecular and Cellular Biology
THE UNIVERSITY OF ARIZONA
MAY 2024

Approved by:
Dr. Walter Q. Betancourt
Department of Environmental ScienceABSTRACTPerhaps the greatest mistake of the scientific community is choosing to exclusively
portray their cutting- edge molecular discoveries in formal, jargon-filled reports. Whileclassic scientific articles have proven to be a very successful enterprise whencommunicating between experts in the field, the average individual does not have thetime nor the energy to sit through a lengthy paper in their free time. To bridge the gap
between academic and non-academic communities and keep the public informed, weneed a more effective way of communicating molecular biology to the masses. The
following Honors Thesis addresses these issues by presenting two years of original
virology research as a molecular biology magazine in order to best display the manydifferent projects at the University of Arizona’s Water & Energy Sustainable Technology(WEST) Center. The magazine itself is centered around a variety of highlyinterdisciplinary projects in waterborne enteric viruses and recycled waters, specificallyemphasizing the improvement of laboratory virus detection methods. The
magazine-style format of the project allows the work to be both comprehensible andengaging for non-scientific audiences while still remaining informative for professionals.
Combining the spheres of art, creative writing, and molecular biology will greatly aidinterest and understanding in complex research topics across a variety of scientificdisciplines.

Abigail Albrightwest.arizona.edu

May 2024

VIRAL
VIGILANCEWATERBORNE
VIRAL
PATHOGENS AND
METHODS OF
DETECTION IN
WASTEWATER
How viruses
survive andthrive in water
environments

Whyresearchers
are studying
your sewage

Where ourwater comesfrom and
where it goesWhatscientists are
doing to keep
our drinking
water safe

1

IN THIS ISSUE...
4

Why Waste?

7

Basic Virology

9

The Wastewater Treatment Process

11

Laboratory Water Analysis

15

Arizona’s WEST Center17

Indicator Virus Research

19

WEST Collaboration with InnovaPrep

21

WEST Collaboration with Yokogawa

23

What’s Next in Water Reuse Research?

25

Resources

30

About the Author & Acknowledgements

2

WHY
WASTE?
Wastewater - that is, everything that comes outof our bathrooms, laundry machines, restaurant
sinks, and farms - is something most people
don’t think twice about.
So, why on earth are scientists so
focused on studying our sewage?

4

Human enteric viruses such as Norovirus,

After the bulk of the disease has passed, theEnterovirus, Astrovirus, and Rotavirus affect thedata obtained from WBE can help estimate

human

by

which intervention strategies were effective andclinging to the walls of the small intestine andwhich were not by cross-referencing dates withusing our own cells to multiply [1, 2]. These

infection levels. WBE has been a crucial tool inviruses and others can be transmitted fromreflecting upon the COVID-19 pandemic, as the

personcontaminateddata collected was used to evaluate thedrinking, bathing, or recreational water use.

efficiency of “stay-at-home” ordinances and re-

Therefore, they are known as waterborne

openings. In the future, the results of Covid-19

enteric viruses, named for their method of

WBE will greatly aid future leaders in theirtransportation and where they are found withinoutbreak responses.

gastrointestinal

topersontract

through[Table1]humans [3]. Recognizing the resilience ofenteric viruses, the human body tries to expel
them instead of killing them. As a result, the
610average sick individual sheds from 106
to 1010virus particles in their waste even if they aren’t
experiencing symptoms. In just one gram of
untreated human waste, there can be hundreds“SARS-CoV-2 is shed inhuman feces up to 6 days
before individuals begin
showing symptoms.”

to billions of virus particles. Waterborne virusesareextremely

difficulttoeliminate

fromwastewater, being resistant to many common

Studying sewage can also help prevent diseasechemicals and water treatment processes [4].

outbreaks from unclean water [5]. As medical

Because waterborne enteric viruses are present

technology skyrockets and life expectancy rises,

in high numbers in our sewage and are difficult

many are questioning if we have enough waterto remove, scientists and data analysts haveto support the resulting levels of population

developed a number of techniques to countinflux. While the amount of water on our earth

these viruses in order to estimate infection rates

never changes due to our closed atmosphere,by taking samples directly from sewage.

global access to safe drinking water is rapidly
declining. There can be hundreds to billions of

Sewage can be used in wastewater-based

virus particles in just one gram of human waste,epidemiology

that

but in many cases, it takes less than 10 particles

measures the number of infected individuals inin water to cause an infection [8, 9]. Evena population in order to plan for and avert

bathing in infected waters can lead to diseasedisease outbreaks [5, 6]. Previous studies have[2]. As of 2022, 2.2 billion people across theshownpathogenworld did not have access to safe and clean

responsible for causing Coronavirus Diseasedrinking water for at least some part of the year2019) is shed in human feces up to 6 days before[10]. As a result, 28% of the world’s population is

individuals begin showing symptoms; otherforcedenteric viruses follow the same pattern. If

contaminated water containing high levels ofscientists constantly monitor common virus

waterborne enteric viruses. Understanding thelevels in community wastewater, they can know

infectivity and survival conditions of the viruses

when an influx of disease is coming andwe shed can help determine the levels of waterintervention strategies can be put in place totreatment needed to prevent environmental

best

contaminationthat

prepare(WBE),SARS-CoV-2

individuals,

policymakers [6, 7].
5atechnique(thehospitals,

and

to

drink

and

and

bathe

protect

communities from epidemics.

inlikelyvulnerable

In the United States, nearly all of our drinking,drought. In states like California and Arizona,tap, and utility water comes from either surface

inconsistentwater or groundwater. Surface water is taken

environmental cleansing methods and access tofrom rivers, streams, reservoirs, or seas - basicallyclean

anywhere there is a body of standing freshwater.

policymakers are reinventing how they recycle

The cleaner of the two, groundwater is hidden

ourunderneath land, rocks, and soil [11]. Once theenvironmental buffers altogether. While it mightwater is pulled from these environmental

sound questionable at first, direct potable reuse

sources, it is treated at a water treatment plant

is a safe way of thoroughly treating our used

for either potable reuse (drinking, shower, andwater and sending it straight back for humantap water) or non-potable reuse (car-washing,use instead of discharging it into bodies ofirrigation, and toilet-flushing). After it is used bywater. Left undisturbed, natural water sources

residences, restaurants, and farms, the dirty

will slowly restore themselves, reestablishingwater travels through underground pipes to ahealthy marine ecosystems and ending ourwastewater treatment facility where it is filteredcenturies-long cycle of human waste dumping

and released back into nearby rivers, streams,

into the natural world. Additionally, drought-

and lakes. The bodies of surface water that serve

prone communities would be able to haveas both a source and a dumping ground are

constant access to clean water, even whenknown as environmental buffers, and their use

surface water flow quality and quantity varies.

ensures that the water is naturally cleansed

Therefore, scientific research on the eradication

before it is taken back to treatment plants forof waterborne enteric viruses in recycled waters

human use. However, this method of indirectcontinues to be a major field in the 21st century,

potable reuse is becoming less and less helpful

especially as we aim to normalize direct potablewithin populations that frequently experience

reuse and provide clean water to all [12, 13].

river

water.
water

As

by

flows
a

put

result,phasingastrainscientists

out

the

use

on
and
of

Table 1: Common waterborne gastrointestinal viruses
Virus

Stability in Wastewater

Size

Adenovirus

Complex virus architecture. Major capsid protein variability and

90 - 100

complexity [24, 25, 26]. Most UV-resistant health-related virus.

nanometers

Stable at pH 3.5. Resistant to heat, ether, chloroform, and mild

27 - 40

detergents [29].

nanometers

Genotype-specific resistance to chlorine [30, 31]. Wide pH

30 - 32

stability range.

nanometers

Can survive in 4-20 degrees Celsius for months. Wide pH

60 - 100

stability range.

nanometers

13
Shed in high numbers (10E3genome
copies per microliter) in

28 - 30

feces. Resistant to pH 3 and high levels of heat.

nanometers

Norovirus

Enterovirus

Rotavirus

Astrovirus

6

VIROLOGY

A crash course in studying viruses
To meet safe water goals, scientists must understand how
human enteric viruses function, inside and out. Like living
things, viruses have an identifying and unique internal
code, called a genome. Genomes are composed of anumber of smaller genes which provide instructions formaking proteins. Humans have tens of thousands of genes
specifying visible traits, food processes, and responses tocellular attack [14]. All of these and more are coded withinhuge strands of deoxyribonucleic acid, or DNA for short.
DNA consists of millions to billions of 4 repeating chemicalgroups called nucleotides, paired to each other andwrapped into a double-helix structure. Although greatly
varying in length and pattern per organism, DNA is foundwithin the cells of every living thing.
In contrast to humans, viruses typically only have a handfulof genes, with the biggest known virus only having 200 [15].
While some viruses have DNA genomes that look likeminuscule versions of our own, many have genomes madeof ribonucleic acid, or RNA, instead. With a slightly different
chemical structure of nucleotides, RNA is less stable thanDNA, and usually only exists in single-stranded form as
opposed to DNA’s iconic double helix. Interestingly, viruses
are the only known thing to carry RNA genomes, making
them unlike anything else on earth [16].
Vastly different from each other on the inside, viruses canalso display a wide variety of external physical features. Allviruses have a protein coat called a capsid that allows for
defense against environmental factors such as extreme
temperatures, abrasive chemicals, and high acidity levels.
Many but not all viruses also have a specialized envelope an additional layer that allows for easier virus replication[17]. Non-enveloped viruses are more difficult to kill, being
able to evade disinfection chemicals much better thantheir enveloped counterparts by clinging to dissolved
material in wastewater [18].
7Upon coming into contact with a desirable cell, viruses
attach, enter, and “hijack” the cell using a unique
combination of surface proteins. Once inside, viruses
force the cell to replicate the virus’ genome instead of its
own, leading to the creation of more identical viruses.
Human enteric viruses cannot replicate without a live
human host cell, meaning that they do not have theability to multiply outside of the human body. This
characteristic is part of what makes viruses notoriously
non-living; their replication requires a non-virus host to
give them access to cellular machinery that they do notcarry in their own bodies. Never alive but surely not dead,viruses still pose a threat because they can lie dormant inwater environments for weeks to months, waiting
patiently for a human cell they can use to replicate.
Because all viruses have unique sizes, combinations ofsurface proteins, and chemical makeups, it is impossible
for one chemical to completely eliminate them all. For
example, chlorine easily kills enveloped viruses, but has
been proven massively unsuccessful in killing nonenveloped viruses [18, 19, 20]. Alternatively, the use ofchemical cleaning agents called aldehydes destroys theinfectivity of nearly all viruses, but cannot be used inwastewater treatment trains due to their hazardous
effects on human health [21]. Viruses are also difficult toeliminate due to their very fast mutation rate, meaningthat their genomes sporadically change in pattern,
making them resistant to reliable disinfecting chemicals.
Human genes also mutate, leading to a wide variety ofevolutionary benefits, diseases, and cancers, but viruses
experience a mutation rate exponentially faster than thatof any other living thing. For example, scientists haverecorded thousands of nucleotide sequence differencesfrom the original SARS-CoV-2 virus since January of
2020, leading to 40 different variants with their own setsof symptoms, resilience, and severities [22, 23]. With theircomplex individuality, waterborne human enteric viruses
require multiple, specialized water treatment trains to be
fully eliminated [Figure 1].
Pictured here, adenoviruses are large, non-enveloped viruses
with double-stranded DNA genomes. A few major capsid
proteins include the penton, hexon, fiber, and core proteins [24].

8THE WASTEWATERTREATMENT PROCESSThe water treatment process can be simplified down to 5 main steps after it is removed fromsurface water and brought to a treatment plant [33]:1

COAGULATION. Chemicals with a positive charge such as iron and aluminum

2
3

FLOCCULATION. The particles of dirt, bacteria, and other dissolved substances

4

FILTRATION. Adsorption-elution strategies use a “media bed” filled withsalts neutralize the charge of negatively charged dissolvents in the water likedirt and sand. This method also neutralizes a few bacteria, algae, and viruses,
but is insufficient in eliminating the majority [34].

begin to clump together in large particles called floc when vigorously mixed
with the above chemicals in a tank.
SEDIMENTATION. Because the floc are heavier and more dense than water,
they settle to the bottom of the tank and do not continue on to future water
treatment steps.

activated charcoal or sand to mimic environmental water cleansing systems.
Ultrafiltration uses membranes with microscopic pores to catch smaller
particles of sediment that might have been missed during coagulation. Even
more precise than ultrafiltration methods, reverse osmosis systems use highpressures to force water through a 0.0001 micron membrane [35]. The
resulting water is clean of dirt and waste, but contains a multitude of
microscopic viruses and bacteria that are too tiny to be filtered out by eventhe smallest of filters.

5

DISINFECTION. Finally, it is crucial to kill any remaining pathogens that are
detrimental to human, animal, and plant health. To do this, treatment plants
add small amounts of chlorine or chloramine - about 4 milligrams per liter ofwater [36]. Another newer but viable option for virus elimination is the use of
UV light, which damages genetic material beyond repair by causing
numerous “breaks” and mutations that the virus cannot survive. Fluoride canbe included to improve oral health, while other chemicals may adjust the pH
to optimize taste. Low levels of chlorine are maintained as the water leaves
the treatment facility so that the miles of pipes between the treatment plant
and local communities remain decontaminated. The resulting low levels ofchlorine and chloramine in potable water are safe for all mammals to
consume, but can be lethal to fish and some plants.
Figure 1: Indirect potable reuse in action

9

water is pulled fromthe environment

T PLAN
N
E
T
TM
A
E
TR
COAGULATION

trash and large particles are filteredout and the water is released backinto surface waters

FLOCCULATIONSEDIMENTATIONFILTRATIONDISINFECTION
safe, clean water is distributed to residential
areas, businesses, and farms= chemicals added
10

THE BEGINNINGS OF
WASTEWATER ANALYSIS:PRIMARY CONCENTRATION
So, how do scientists test for and quantify waterborne virus levels?
Laboratory technologies for measuring and

The VIRADEL method, which is based on theconcentrating waterborne viruses have greatly

adsorption of viruses to electro-positively or

improved within the last twenty years, allowing

electro-negatively charged filters, is anotherscientists to accurately perform WBE and ensure

viable option for primary

proper water treatment trains are working.

viruses. The most commonly used filters forconcentration oflarge volumes of water are the positively
Overall, it has long been recognized that the

charged Zeta Plus 1MDS and NanoCeram, both

best methods to assess the occurrence of viruses

components of the United States Environmental

in water will be those that are simple, rapid,

Protection Agency’s (USEPA) Method 1615. The

inexpensive, and consistent. While a number of

EPA method was developed to provide atechniques have been developed and refined, it

standardized

approachformeasuringhas proven difficult to achieve detection of all

waterborne

enteroviruses

andnoroviruses,

relevant virus types over the wide spectrum ofwhich both occur very frequently in the U.S. andwater quality diversity reflected in both natureserve as indicators of water purity [42].

and human-constructed facilities [37]. Because
viruses are present in treated wastewater at very

The use of NanoCeram filters has proven to be

low concentrations, large volumes of 100 – 1,000

both practical and efficient for virus recovery

liters are required for processing to enhanceand concentration from 1,000 to 2,000 liters ofrecovery and detection [37, 38, 39].

highly treated wastewater [43, 44]. Collection ofsuch a large volume of water allows a wide

Hollow fiber ultrafiltration, which relies on

range and quantity of viruses to be detected

excluding certain particles by size, can be usedfrom just one sample. While both hollow fiber

as a viral primary concentration technique [40].

ultrafiltration and VIRADEL technologies have

Ultrafiltration membranes are constructed frombeen recently reviewed [45], many other newer

a variety of polymers such as polysulfone,strategies

likepolyacrylonitrile, and cellulose triacetate, andtangential

flow

can be used in either tangential (cross) flow

significantly

mode or direct (dead-end) flow mode [41].

comparison of these techniques with the EPA

However, ultrafilters are impractical for fieldstandards may provide vital insights into thesampling or processing of extra turbid water, as

ideal combination of methods for targetingtheyelimination of specific waterborne viruses ineasilysediments.

11

becomecloggedwithlargerpassivelessrecycled water.

sampling

filtration

[46]

and

have

been

Analysis

and

[47]

explored.

Afterprimary

concentration,

secondaryconcentration of viruses is crucial for detection
and quantification. Traditionally, this process
involves centricons, the tried-and-true method
used to bring a sample’s volume from a large

FROM LITERS TO DROPS:SECONDARYCONCENTRATION

amount to just a few drops without losing thehigh amount of viruses it contains. A centriconconsists of a top section where a wastewater
sample is placed and funneled through a filter
with microscopic pores upon centrifugation.
Water molecules are small enough to flow
through the filter, while viruses are caught on
the membrane. After all the water is filtered, theMillipore-membrane is inverted and the viruses are

Sigmacollected with a miniscule amount of clean

Centricon

water running in the opposite direction.
An emerging alternative method to centricons is
the InnovaPrep Concentrating Pipette (CP). CPs
boast a number of advantages over centricons,
the first being the amount of sample it can filter
at once. Centricons’ maximum load is 70
milliliters (mL) of sample, which is about ⅓ of acup. InnovaPrep’s CPs can efficiently filter up to 1
liter in just one run, nearly 15 times the amountof a standard centricon. Time also favors CPs.
Centricons require a pre-wash with water and a
30-minute filtering cycle for the wastewater

Separates into a
filtration unit
and a catchsample, making the entire ordeal nearly an hour.
CPs filter their samples in just 5 to 10 minutes,
allowing scientists to capture the same virusesin a much shorter time span.
While they are a convenient and standardized
practice,

centricons

fall

short

in

reliably

capturing every virus equally. Perhaps the mostinnovative feature about the CP is that it offers
the flexibility to incorporate any liquid wash
buffer before and after the uptake of awastewater sample in order to purify viruses as
they arecaptured. This is not possible withcentricons, as water is the only liquid that canflow across a centricon membrane.

InnovaPrep
Concentrating Pipette
12After a wastewater sample is concentrated through either centricons or CPs,
order to determine how prevalent the virus is in a given population. To complete

belonging to a complete viral particle that could interfere with theresearchers’ results. Performing this nuclease treatment ensures that only
viral genomes within intact capsids (and therefore, only full viruses) arecounted. Following nuclease treatment, DNA/RNA extraction uses a seriesof detergents to dissolve the virus’ envelope and/or the capsid. Once theviruses have broken open, repeated centrifugation and filtration remove
any extra proteins or lipids that may be present in the sample, leaving only

Finally, the last step in wastewater virus analysis is quantification andidentification of the virus genetic material. Polymerase chain reaction,abbreviated as PCR, is a classic molecular biology technique first
introduced in the 1980’s to amplify DNA sequences using florescentprobes. Since their invention, PCR methods have expanded, featuring
many variations such as Real Time Quantitative PCR (RT-qPCR) anddigital PCR (dPCR). Analyzing samples by RT-qPCR results in a series ofcurves, which can be compared with the curves of known viralconcentrations called standards to determine the concentration of
the virus within the sample. This method is called relative
quantification, as the interpretation of results depends heavily oncomparison with the standards. Alternatively, dPCR relies on absolute
quantification, which needs no standards because each tiny drop is
split into 24,000 microscopic pieces and analyzed individually. The
results of such an experiment manifest in two distinct clusters, where

From start to finish:

the purified DNA/RNA [48].

the drops containing no viruses show no florescence and the drops
containing high amounts of viruses show high amounts of florescence[Figure 2]. While the two PCR methods differ in sensitivity, chemicals
required, and level of technological complexity, they both perform thesame purpose: accurately replicate, then count the number of virus
genomes in a sample by connecting desired genetic sequences toflorescent probes that are unique for each virus [49].

By using all of these tools, researchers can determine theidentity and amount of viruses within our wastewater.
13

THsample. Nucleases “chew up” any additional free-floating DNA or RNA not

L
L
U
EF

Purification begins with the addition of enzymes called nucleases to the

TER ANALYS
A
W
E
T
S
IS P
WA

ROC

this task, scientists’ must isolate the DNA or RNA inside the virus.

ESS

scientists need to identify and count the virus particles present in a sample inFigure 2

filter from wastewater source

primary concentration

centricons

OR

InnovaPrep

nuclease treatment

DNA/RNA extraction

Real-time PCR

relative quantification

OR

digital PCR

absolute quantification

14

WELCOME TO THE

WEST CENTER

The University of Arizona is ranked number two
in the nation for water resources research,making Arizona’s Water & Energy Sustainable
Technology (WEST) Center a leading venue forincredible molecular environmental studies
with widespread applications. Arising from apartnership

with

Pima

CountyRegional

Wastewater Reclamation Department, WEST’slaboratory doors opened in spring of 2016 witha primary goal to ensure the future of water
sustainability across the Sonoran desert andbeyond.

Withpublichealthinmind,researchers work relentlessly to guarantee thesafety of recycled waters for humans andmarine ecosystems alike. Professionals from anumber of different fields work collaboratively
on projects at WEST, including environmental
scientists, engineers, and microbiologists. In thepast few years, WEST scientists have also joined
together in research with the Centers for
Disease Control and Prevention, United States
Army

Corps

of

Engineers,

Environmental

Protection Agency, US-Israel BARD, California
Water Board, Water Research Foundation,
Arizona Department of Agriculture, Yokogawa
Electric Corporation Japan, and Innovaprep
LLC. Additionally, the WEST Center played amajor role in wastewater surveillance for SARSCoV-2 during the early phases of the pandemic,
tracking the pathogen in wastewater to ensure
the safety of the 40,000+ Arizona students
living and learning on campus. A number ofthrilling projects are always happening at
WEST, all of which are sure to prove massively
beneficial within the field and for the bigger
picture of water sustainability.

16

INDICATOR
VIRUSES TOCONFIRMADVANCED
PHYSICALTREATMENT

Treatment of wastewater for potable reuserequires the reduction of enteric viruses to a
threshold that poses no hazard to human health.
These acceptable limits are different for every
virus, depending on their unique infectivity andassociatedhealthrisks.

Advancedwater

treatment trains have been developed in order tobring viruses down to safe levels, such as
chemicalclarification,

reverseosmosis,ultrafiltration, and advanced oxidation, to name afew [50].
Various studies have evaluated the elimination ofvirusesbydifferent

municipal

wastewaterAn evaluation of water treatment trains

treatment processes in the U.S. and havecompleted by researchers at the WEST Center

concluded that fully treated water still contains
both infectious and noninfectious viruses, posing
great health risks depending on the size of theviral breakout and whether the water is forpotable or non-potable reuse [51-61]. Intrigued bythese previous studies, researchers at WEST
evaluated the reduction of fifteen virus indicators
to confirm advanced physical treatment for

ultrafiltration-

indirect potable reuse. The viruses investigated inreverse osmosis

this study represented extremes in the biologicalpotable reuse scheme

size spectrum, ranging from 15 – 25 nanometers
to 70 – 90 nanometers in diameter. The study also
evaluatedviruseswithvarying

distinctive

genome features, such as single-stranded DNA
viruses, double-stranded DNA viruses, singlestranded RNA viruses, and double-stranded RNA
carbon-based

viruses. The fifteen viruses selected for the

potable reuse scheme

experiments also had an extensive record ofresistance to chemical and physical disinfectants
in wastewater treatment processes. Full-scaletesting involved water reuse facilities in Arizona,
Colorado, and Virginia to represent treatment
plants located in three distinct geographicalregionsofthecontinental

UnitedStates:Southwest, West, and Southeast. Advanced“Fully treated water still contains both
infectious and noninfectious viruses.”treatment trains employed both membranebased(ultrafiltration-reverse

osmosis)andcarbon-based (ozone-biological filters-granulated
activated carbon) potable reuse schemes, plus anadvanced managed aquifer recharge system

17

with soil-aquifer treatment.

The results of the study indicated that a
multitude of factors determine average virus
occurrence

andreduction.

One

major

component is the preexisting virus abundance insourcewaters

upstreamoftheadvancedtreatment trains. Some treatment processes do
not account for contamination and pollution oflakes, rivers, and streams on top of the alreadycontaminated wastewater; in short, they wrongly“Viruses of publichealth significancesuch as human entericviruses were proposedas the best viralindicators to confirm
water purity.”

assume the source waters are always very clean.
Another factor was the

structural differences

between the viruses, which are highly linked tovaryingvirus

resilience.

Differences

in

the

detection method and each treatment plants’operational conditions also played a major role indetermining the concentration of viruses inwastewater. Considering all of these aspects,
viruses of public health significance such as
human enteric viruses were proposed as the best
viral indicators to confirm water purity. Moving
forward, the study suggested that continuous
monitoring of waterborne human enteric viruses
is fundamental to confirming the functionality
and consistency of potable reuse schemes.
Our knowledge on pathogen occurrence andpersistence in the environment has substantially
increased with the development of more efficientand sophisticated technologies for pathogen
detection. Site-specific environmental conditions,the cultural dynamic of local human populations,and viral evolution must be considered in orderto understand virus presence and physicalremoval by advanced water treatment processes.
Equally important is the application of the best
available technologies to meet safe standards forhuman enteric viruses. All of these factors must
be considered when expanding the research
agenda to ensure current and future sustainable,(Farkas et. al, 2020)

reliable, and safe water reuse.

18

WEST COLLABORATION WITH INNOVAPREP

treated wastewater spiked with adenovirus, coxsackievirus,
human coronavirus 229E, murine norovirus, and ORF virus

The sample is divided into 9 completely identical parts.
HOWEVER, each receives a unique combination of wash buffer and software selection
when run through the InnovaPrep Concentrating Pipette.

InnovaPrep software 1.5.0.0 with Tris/EDTA + Tween wash buffer

InnovaPrep software 1.5.0.0 with Tris/EDTA wash buffer

InnovaPrep software 1.5.0.0 with NAPP + Tween buffer

InnovaPrep software 1.5.0.0 with no wash buffer

InnovaPrep software 6.3.2.1 with Tris/EDTA + Tween wash buffer

InnovaPrep software 6.3.2.1 with Tris/EDTA wash buffer

InnovaPrep software 6.3.2.1 with no wash buffer

Centricons with water wash

Processed right after filtration as a negative control
19One major subdivision of research at the WEST Center is in developing
alternative methods of secondary concentration to make virus detection
faster, simpler, and more accurate. Researchers at the WEST Center
Figure 3

constructed a series of experiments to compare centricon and CP
performance for secondary concentration of waterborne enteric viruses.
In these experiments, researchers added predetermined amounts of viruses toa highly treated sample of tertiary treated effluent. The wastewater sample
received high amounts of adenovirus, coxsackievirus, human coronavirus
229E, murine norovirus, and ORF virus in a process called cocktail spiking. The
team then concentrated nine identical cocktail spiked samples using ninedifferent methods, including by centricons and by CP with unique buffer andsoftware combinations. After concentration, the samples were all nucleasetreated and extracted in an identical manner, and the resulting variations
were quantified by RT-qPCR. The protocol detailed in Figure 3 allowed
researchers to quantitatively rank the efficiency of the different methods foreach virus by observing the differences in PCR amplification levels from thetrue amount added during cocktail spiking.
The results of this study strongly suggest that a single protocol for viralconcentration cannot possibly account for the wide variety of waterborne

Each sample
is analyzed
for all virusesby RT-qPCR

enteric viruses, as the same spiked sample resulted in vastly different
quantification results depending on the sample concentration method
employed and the virus being tested. For example, using a CP with 25 mM Tris
+ 1 mM EDTA + 0.1% Tween wash buffer allowed for 60 to 70% recovery of
adenoviruses, whereas other methods only allowed for 20%, signifying thatthe former buffer is the most accurate method to capture adenoviruses. By
contrast, crAssphage, an endogenous wastewater virus, was recovered bestwhen washing steps were not used at all. Additionally, human coronavirus
229E benefited most in procedures that didn’t include the CP whatsoever(such as centricons). Early data also suggests that the use of Tween buffers
inactivates enveloped viruses, as we continue to see significantly less thanspiked concentrations in any protocol where Tween is used. This is apotentially dangerous observation, because any added chemical that disrupts
a virus could be leading to inaccurate PCR results and a false sense of security
when testing wastewater and receiving low virus counts.
Ongoing collaborations between WEST Center scientists and InnovaPrep LLC
are focused on repeating these studies to confirm the most efficient approach
for individualized testing according to each specific virus size, charge, andgenome makeup. Once a standard protocol has been developed, waterborne
enteric virus recovery efficiencies are sure to significantly improve.

20

WEST COLLABORATION WITH YOKOGAWA

Filipić, 2021

pepper mild mottle virus (PMMoV)
Known to affect peppers, PMMoV is the most abundant RNA found in human feces. PMMoV
is very stable in wastewater, and though it is not a human enteric virus, it can serve as afantastic indicator of water cleanliness due to its year-round global occurrence.

21

To solve a number of challenges with consistency

Extractionof wastewater samples in the lab, a technology

important step in making sure PCR only counts

that can directly and quickly detect viruses iswhole virus genomes. The most frequently useddesired.

astechnique makes use of a number of detergents,

VIRADEL can achieve these goals, researchers

chemicals, and filters to achieve extracted

and engineers hope to continue improving upon

genetic material. However, the RAPID techniquethese methods to make them even more

introduces a newly engineered process which

accurate and time efficient. In a partnership withwas hypothesized to save time and energy while

Yokogawa

still

While

current

Electric

protocols

Corporation

suchJapan,

the

of

viral

genetic

maintaining

material

optimalisanperformance.

University of Arizona WEST Center aimed to

Yokogawa’s high temperature pressure (HTP)

validatepathogenextraction method heats and pressurizes aforsample for one minute in order to completely

anidentification

integrated

rapid(“RAPID”)

systemtheassessment of membrane integrity.

extract the viral nucleic acid material. More than
40 times quicker than traditional reagent-based

Yokogawa’s RAPID method consists of three

extraction methods, HTP provides an interesting

components...

and new approach to sample processing. As partof the validation of Yokogawa’s RAPID technique,
WEST researchers evaluated the efficiency of HTP

1concentration of viruses

2

viral nucleic acid extraction using aproprietary high temperaturepressure (HTP) extraction method3detection and quantification of viralnucleic acid using RAPID-based
molecular amplification

extraction methods as compared to chemical
extraction methods for Adenovirus, two plantviruses (Pepper mild mottle virus and Cucumber
green

mottlemosaicwastewater-associatedvirus),

Aichi

virus,

CRESS

viruses,andcoliphages. Interestingly, HTP extraction seemed
to work just as well as chemical extraction whenanalyzedby

RT-qPCRbut

severely

underperformed when using digital PCR. This
collaborative study showed that faster and newer
methods don’t always work for every virus,
emphasizing the importance of individualized
protocoldesignfor

each

desiredvirus

quantification.
The Yokogawa project is ongoing as of spring of
2024. Within the next year, risk assessment
approaches will be used to identify reference
pathogen reductions needed to meet current log
credit allocations in the United States and to
estimate infection risks associated with the
RAPID system as opposed to the VIRADEL
system.
Model recreation of Aichi virus structure

22

WHAT’S NEXT?
Future studies happening at WEST
Moving forward, frequent testing of highly
treated wastewater for human enteric virus
indicators is necessary in order to make surethatwastewater

treatment

plans

are

working. Breakthroughs of indicator viruses
such as adenovirus or enterovirus can signal
a problem with the water treatment process
that

might

not

be

detected

without

appropriate sampling monitoring schemes
and methods. Further studies will continue
monitoring indicator viruses using different
primary and secondary virus concentration
approaches. In addition, viral metagenomics
will be applied in order to identify key
patterns of virus genetic sequences that areassociated with treatment resistance.
Another emerging branch of research at
WEST concerns other harmful agents inwater besides pathogens, like lingering
agriculturalchemicals,

microplastics,orpolyfluoroalkyl substances (PFAs). All of theabove have proved to be damaging tohuman health and are particularly difficult
to remove from water, which warrants
serious consideration on how to efficiently
eliminate them from our recycled waters.

23

Whether you are aware or not, all of the water you drink has been
recycled through other humans and the environment for centuries.
Rivers are becoming less reliable, their flow varying greatly with thechanging seasons. When millions of people are dependent on thoseriver flows in order to bring them safe freshwater to serve as anenvironmental buffer in indirect potable reuse, a drought can havesevere consequences. Bypassing our rivers by direct potable waterreuse can solve this problem, and is already approved in two cities in
Texas and on a case-by-case basis in Arizona. As of late 2023,
Californians also frequently drink highly cleaned and treated recycledwater without the use of an environmental buffer. In these early yearsof direct potable reuse, extensive, continued water resources research is
absolutely necessary. If current treatment methods are allowing forviral breakthroughs - even if the breakthroughs are small - better
technologies need to be developed.

"Progress in science depends on
new techniques,
new discoveries,
and new ideas,
probably in that order."-Sydney Brenner

Performing research on waterborne enteric viruses and water
treatment processes allows scientists and engineers to makeinnovative changes to wastewater analysis protocols worldwide. Only
by improving our techniques can we collect crucial data about disease,make safe drinking water accessible, and protect public health.

24

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Photographs:[pg 7-8] Cancer Histology Core. (n.d.). Pathbio.med.upenn.edu. https://pathbio.med.upenn.edu/pbr/portal/hist/F19adeno.html[pg 12] MilliporeSigma Centricon Plus-70 Centrifugal Filters - Filters and Filtration, Centrifugal Filter Devices. (n.d.).
Www.fishersci.com. Retrieved April 26, 2024, from https://www.fishersci.com/shop/products/emd-milliporecentricon-plus-70-centrifugal-filter-units-3/p-3081951[pg 17] 500L RO Water Treatment Equipment for Directly Drinking Water. (n.d.). Made-In-China.com. Retrieved April
26, 2024, from https://xajufu.en.made-in-china.com/product/unWRVQkGsXch/China-500L-RO-Water-TreatmentEquipment-for-Directly-Drinking-Water.html[pg 17] Support, A. (2017, June 4). Is There a Difference Between Activated Carbon & Charcoal Water Filters? Guardian
Water Services. https://www.guardianwaterservices.com/activated-carbon-vs-charcoal-water-filters/[pg 18] Farkas, K., Walker, D. I., Adriaenssens, E. M., McDonald, J. E., Hillary, L. S., Malham, S. K., & Jones, D. L. (2020).
Viral indicators for tracking domestic wastewater contamination in the aquatic environment. Water Research, 181,115926.
[pg 21] Filipić, A., Dobnik, D., Tušek Žnidarič, M., Žegura, B., Štern, A., Primc, G., Mozetič, M., Ravnikar, M., Žel, J., &Gutierrez Aguirre, I. (2021). Inactivation of Pepper Mild Mottle Virus in Water by Cold Atmospheric Plasma. Frontiersin Microbiology, 12.
[pg 22] Charles David Sabin, Tibor Füzik, Karel Škubník, Lenka Pálková, A. Michael Lindberg, & Pavel Plevka. (2016).
Structure of Aichi Virus 1 and Its Empty Particle: Clues to Kobuvirus Genome Release Mechanism. Journal of Virology,
90(23), 10800–10810. https://doi.org/10.1128/jvi.01601-16

All other photographs are available on their respective product websites (WEST Center, InnovaPrep, MilliporeSigma,
Qiagen, and Roche). Any other photos [pg 15-16] taken by Abigail Albright.
Magazine created with copyright-free Canva design tools.

29

ABOUT THE AUTHOR
Growing up outside of the scientific community, Abigail
found herself disinterested in research, mainly because
she could never understand it. Unfortunately, cuttingedge molecular discoveries seem to take one of two
forms: a flashy, usually misleading headline, OR alengthy, jargon-filled report. While formal papers arehelpful in communicating between experts in the field,
the vast majority of people have neither the interest nor
the time to decode a twenty-something page article.
Her hope is that this magazine will play a part inbreaking down the walls that isolate the scientificcommunity,EP

science

accessible

andunderstandable for all.

L T H ANA
I
C
Walter Q. Betancourt, PhD
Aidan Foster, PhD
Kaitlin Chung

KS

S

Abigail AlbrightmakingJustin Clark
Leandro Phelps-Garcia
Lois Polashenski
Yoneri Bueno-DiazSkyler Tilden
WEST Center Staff and Students
The University of Arizona
Arizona Department of Agriculture
California Water Board
Centers for Disease Control and Prevention
Environmental Protection Agency
InnovaPrep LLC
United States Army Corps of Engineers
US-Israel BARD
Water Research Foundation
Yokogawa Electric Corporation Japan
30