mm mm®©mn(§WK] m

G, Shay Fout1, Ann C. Grirnm1, Daniei R. Dahling1, Jennifer L. Cashdollar1, Christina M, Newport1, Sandhya Parshionikar2, Sandra Wiiiian-True2
1 National Exposure Research Laboratory, Cincinnati, Ohio; 20akridge Postdoctoral Trainees, Cincinnati, Ohio

Emerging	Tec

Environmental Issue:

WATERBORNE DISEASE (1920-2000)

350
300
250
200
150
100
50
0

OUTBREAKS PER DECADE

1920s 1930s 1940s 1950s 1960s 1970s 1980s 1990s 2000s

¦	Waterborne disease outbreaks occur every year

¦Annual average is 23 outbreaks with 19,000 cases
¦Over half of the outbreaks are caused by viruses

¦	The actual disease burden from waterborne viruses is unknown
because of a lack of detection methods.

Collaborative Approach:

A partnership of scientists and engineers from ORD, the Office of Ground Water and Drinking Water's
Technical Support Center (TSC) and the American Water Works Association Research Foundation
(AWWARF) was formed to improve every aspect of standard virus sampling procedures and to develop
a new molecular virus detection method. The steps that are needed to detect viruses in water are as
follows:

1. Sample Collection:

Alignment of Enterovirus
RNA Sequences

The emerging
tools of
molecular
genomics and
bioinfomatics
was used to
develop the
new method.

2. Filter Processing

Results

Ground Water:

The virus method developed under the partnership
between ORD, TSC and AWWARF was used to examine
29 ground water wells on a monthly basis for 12 months.
Results showed that 16% of 321 groundwater samples
and 72% of 29 groundwater sites were positive for
viruses.

Virus in water are collected on filters

Viruses are removed from filters with
over 90% recovery.

3. Virus Purification

4. Virus Detection

RT - PCR Analysis of Samples
1 234 567

BP

Virus are purified and concentrated over
500,000 times with greater than a
70% recovery.

A new method was
developed to detect major
groups of waterborne
viruses.

RESULTS

Virns

% Positive
Samples Sites

Enteroviruses

5

38

Reo virus

10

62

Rotaviruses

0

0

Hepatitis A virus

1

14

Norwalk virus

3

21

Total

16

72



Surface Water:

A second partnership was developed between ORD and
the U.S. Geological Survey to test the methods using
stream waters from five rural to urban sites across the
U.S. The method proved effective for surface water with
all sites being positive for viruses as shown below.

Rotavirus
Hepatitis A virus
NorwaLk virus

Hepatitis A virus
Poliovirus

Site

Land Population
Use Density3

Percent Virus
Enterovirus

Positive Samples
Rotavirus HAV

EIWA

Rural" 16

100

0 0

KANA
SANT

Rural" 88
Rurald 544

33
100

0 0
100 33

LERI

Urban6 2849

100

67 67

PUGT

Urban 15540

100

33 0

1 Agricultur

a Agricultu
' Site includ

per square kilometer
and confined animal facilities
as and septic tanks near site

es some agricultural contribution

mm



Outbreaks:

A third partnership was developed between ORD, EPA
Region VIII, CDC and state Departments of Health to
investigate two groundwater-related outbreaks of
disease in the state of Wyoming in 2001. The method
proved valuable for outbreak investigations in that
norovirus strains were detected in the groundwater and
shown to be identical to patient strains. The two
outbreaks were caused by different norovirus gene
groups.

Calicivirus

Viruses in Group:

Norovirus

Genogroup I
Norwalk
Genogroup II
Snow Mountain

Nucleic Acid:

Single-Stranded

Disease:

Gastroenteritis

Impact:

¦	The virus detection method is available to collect virus
occurrence data needed to support the development of
regulations to protect the American public.

¦	The virus detection method is available to support
exposure studies designed to determine the disease
burden caused by waterborne viruses.

¦	Data from using the virus detection method will lead
to improvement of drinking and recreational water
quality.

¦	The method is adaptable to testing for new viruses in
water, such as the recently emerging SARS corona-
virus.

Partnering	to Protect	Human


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