United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-83-011 Aug. 1983
&EPA Project Summary
Studies of Waterborne
Agents of Viral
Gastroenteritis
Ralphael Dolin
The etiologic agent of a large outbreak
of waterborne viral gastroenteritis was
detected employing immune electron
microscopy (IEM) and a newly devel-
oped solid phase radioimmunoassay
(RIA). This agent, referred to as the
Snow Mountain Agent (SMA). is 27-32
nm in diameter, has cubic symmetry,
and is morphologically similar to but
antigenically distinct from the previous-
ly described Norwalk and Hawaii agents.
After an incubation period of 18 to 48
hours, the agent is shed in stools of
acutely ill individuals for a period of one
to five days.
Employing both IEM and RIA, serum
antibody rises were observed in 3/3
naturally-occurring cases and in 8/9
cases of experimentally-induced illness
in normal volunteers. IEM and RIA
appeared to be equally sensitive for
detection of antibody rises, but the RIA
was more sensitive than IEM for the
detection of SMA in stool specimens.
Preliminary attempts to cultivate the
agent in vitro were unsuccessful.
The development of the RIA for SMA
provides a sensitive and efficient meth-
od with which to assess the epidemio-
logic significance of this agent and with
which to begin evaluation of potential
control measures.
This Project Summary was developed
by EPA's Health Effects Research Labo-
ratory, Research Triangle Park, NC, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Objectives
Viral gastroenteritis is a major public
health problem for which there are as yet
no adequate control measures. The devel-
opment of control measures has been
hindered by the lack of information con-
cerning the specific etiology of many
cases and by the poor understanding of
the transmission and epidemiology of this
disease. In December of 1976, a large
outbreak of acute gastroenteritis occurred
at a resort (Snow Mountain) near Granby,
Colorado. The characteristics of illness
were typical of viral gastroenteritis, name-
ly fever, vomiting, and diarrhea which
lasted 24 to 48 hours. The attack rate was
high (418/762 individuals at risk) and
person-to-person spread was noted. Ill-
ness was associated with consumption of
water- or ice-containing beverages in a
dose response manner (p<.0001). Stool
specimens were negative for bacterial
pathogens, and virus cultures employing
standard tissue culture systems were
similarly negative. The current studies
were carried out to investigate the etio-
logy of this outbreak and to develop
sensitive and efficient methods for the
detection of the causative agent.
Methods
Because of the suspicion that a non-
cultivatable viral agent was likely to be
involved, the techniques employed were
those that have been used successfully to
study the Norwalk agent of viral gastro-
enteritis. Five stool specimens from
acutely ill individuals in the Snow Moun-
tain outbreak were examined by immune
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electron microscopy employing pooled
gamma globulin as a source of antibody.
Twenty-seven to 32 nm virus particles
were detected in two of the samples.
Because the amount of material available
from the naturally-occurring outbreak
was limited, a series of studies in normal
volunteers was undertaken in which
stool filtrates containing the above virus
particles were administered to normal
volunteers. Gastrointestinal illness was
induced in nine of twelve volunteers.
Stools and sera from the volunteer study
were examined by immune electron
microscopy, employing 2% stool filtrates
and post-challenge serum specimens.
Immune aggregation of the particles, in
which clumps of virus were coated by
specific antibody, permitted identification
of the agent in stool suspensions.
With the use of high titered serum
specimens and concentrated stool prepa-
rations identified by immune electron
microscopy, a solid phase radioimmuno-
assay was developed. This assay depends
on the differential binding of antigen to
pre- and post-challenge sera from cases
of experimentally-induced illness after
challenge with an infectious inoculum.
The assay can also be used in a blocking
test to detect antibody to the agent being
sought.
Results
Examination of stool filtrates from
volunteers challenged with SMA by im-
mune electron microscopy revealed 27-
32 nm particles in stools from three
acutely ill volunteers. Particles had cubic
symmetry and were morphologically distin-
guishable from Norwalk and Hawaii
agents. With the use of one such filtrate
as a source of antigen, serum antibody
rises were detected in eight of nine
volunteers with experimentally-induced
illness and in three of three naturally-
occurring cases. Virus was shed by
volunteers 24-48 hours after challenge,
generally at the height of illness.
With the use of high titered sera and
concentrated stools as determined by
immune electron microscopy, a solid
phase radioimmunoassay was estab-
lished for the Snow Mountain Agent. This
assay detected virus that was shed over a
time period similar to that detected by
IEM but appeared to be shed for as long as
five days in some volunteers. The radio-
immunoassay detected the Snow Moun-
tain Agent in all stools that were positive
by IEM and in a number of IEM negative
stools as well. Serum antibody rises were
seen in eight of nine ill volunteers chal-
lenged with the Snow Mountain Agent
and m three of three naturally-occurring
cases. Volunteers who were challenged
with SMA but did not become ill did not
manifest antibody rises by either IEM or
RIA. Preliminary attempts to cultivate the
Snow Mountain Agent//? vitro in standard
tissue culture systems have been nega-
tive.
Conclusions
• The etiologic agent of the Snow
Mountain outbreak is a 27-32 nm
virus that is cubic in symmetry and
morphologically indistinguishable
from the Norwalk and Hawaii agents.
• The Snow Mountain Agent (SMA)
can be detected by immune electron
microscopy and is shed by infected
volunteers during acute illness.
• A highly specific radioimmunoassay
for Snow Mountain Agent was devel-
oped that is more sensitive and more
efficient in the detection of SMA than
immune electron microscopy.
• Both immune electron microscopy
and the solid phase radioimmuno-
assay can be used to detect serum
antibody rises which occur in high
frequency in naturally-infected and
experimentally-induced disease.
• Snow Mountain Agent appears to be
antigenically distinct from Norwalk
and Hawaii agents both by immune
electron microscopy and radioim-
munoassay.
• The radioimmunoassay appears to
be the method of choice for detection
of both SMA antigen and anti-SMA
antibody.
Recommendations
Development of a radioimmunoassay
for the Snow Mountain Agent provides a
sensitive yet efficient method with which
to conduct additional studies of SMA.
Specific recommendations for studies to
be conducted are as follows:
1. assessment of the frequency of infec-
tion with SMA in sporadic and epi-
demic forms of acute viral gastroen-
teritis, particularly those in which a
waterborne or common source has
been identified,
2. description of the seroepidemiology
of the Snow Mountain Agent employ-
ing age, socioeconomic and geo-
graphically distributed populations,
3. sampling of environmental sources,
particularly water supplies,
4. intensification of efforts to develop
suitable in vitro cultivation systems
employing the radioimmunoassay
as a method to detect viral growth,
5. because SMA and other viral agents
of gastroenteritis are known to under-
go waterborne spread, evaluation of
the effectiveness of decontamination
procedures upon water supplies as a
potential control measure.
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Ralphael Dolin is with the University of Vermont College of Medicine, Burlington,
VT 05405.
Elmer W. Akin is the EPA Project Officer (see below).
The complete report, entitled "Studies of Waterborne Agents of Viral Gastro-
enteritis, " (Order No. PB 83-234 930; Cost: $8.50, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
ftUS GOVERNMENT PRINTING OFFICE 1983-659-017/7156
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