United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 2771 1
Research and Development
EPA-600/S1-83-004 June 1983
&EPA Project Summary
Determination of Minimal
Infectious Dose of an Enterovirus
in Drinking Water
Gilbert M. Schiff, Gerda M. Stefanovic, Betsy Young, and Julia K. Pennekamp
The goals of this project were to
determine the minimal infectious dose
and the medical significance of an
enteric virus (Echovirus-12) ingested
in drinking water. The study was con-
ducted under double-blind, placebo-
controlled, random-selection conditions.
A total of 149 susceptible (antibody-
free), healthy, young adult males were
placed in isolation and ingested varying
amounts of Echovirus-12 (10-10,000
pfu) or placebo seeded into 100 ml of
nonchlorinated water. The subjects
were followed for evidence of illness,
pharyngeal and intestinal (fecal) viral
shedding, and serum antibody response.
No illness or evidence of infection
occurred among the placebo subjects.
Infection in exposed subjects was pri-
marily based on fecal shedding of virus.
Only four subjects experienced a sig-
nificant seroconversion in the absence
of demonstrable fecal viral shedding.
Similarly, pharyngeal shedding of virus
without evidence for fecal viral shed-
ding was sporadic and was observed in
only five subjects. Fecal shedding
and/or significant seroconversion oc-
curred in 30% of the subjects given 10
pfu (the lowest dose administered)
and in 100% of the 12 subjects given
300 pfu as measured in a plaque assay
using LLC-MK2 cells. Fecal shedding
persisted for as long as four weeks in
some subjects.
Statistically, it was inferred from
probit analysis that ingestion of one to
two pfu of Echovirus-12 would infect
1% of the population.
This Project Summary was developed
by EPA's Health Effects Research Lab-
oratory, 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
Background
Waterborne outbreaks of viral disease
continue to occur. Viruses which are
known or highly suspected to contaminate
untreated and/or treated waters are hepa-
titis A virus (now tentatively classified as
an enterovirus), enteroviruses, adenovi-
ruses, and the group of nonbacterial gas-
troenteritis viruses (rotaviruses, Norwalk
agent, and others). Current technology is
capable of detection of small quantities of
viruses in relatively large amounts of water.
Because enteroviruses are known to oc-
casionally survive water treatment, they
are a potential hazard to human health.
What is not known is the medical signifi-
cance of small amounts.of viral contamina-
tion which may occur in potable waters.
For economic and scientific reasons,
there is no widespread program for moni-
toring viruses in drinking water, and very
little data are available about health effects
of low-level viral contamination of water
supplies. This project was undertaken to
demonstrate the critical level of water
contamination which produces an infec-
tion of illness in humans. The results
would provide a basis for more informed
decisions regarding the establishment of
standards for viral content of treated
waters.
To determine this critical level, experi-
mental viral "challenge" studies were con-
ducted under rigidly controlled conditions.
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Susceptible subjects were chosen, known
doses of a human virus were administered
at recorded times, and subjects were care-
fully observed and monitored using lab-
oratory sampling procedures. The research
team used a viral representative found in
water, and administered this virus to sub-
jects in drinking water. During its use in a
previous experimental viral challenge study,
this virus had not been associated with
serious human illness.
Specific Goals
The specific goals of the study were to
determine in susceptible, healthy, adult
subjects: (1) the minimal infectious dose
of Ediovirus-12 ingested in drinking water
and (2) the medical significance of small
amounts of an enteric virus consumed in
this manner.
Study Design
The study was designed to select healthy,
young, adult male subjects who lacked
detectable serum hemagglutination-inhi-
bition (HI) antibodies to Echovirus-12,
place them in isolation, inoculate them
with varying doses of the vims (10-10,000
pfu assayed in LLC-MK2 cells) or placebo
(tissue culture fluid) and follow them for
evidence of illness, viral shedding in the
pharynx and intestinal tract, and produc-
tion of serum antibody. The assignment of
inoculum (viral dose or placebo) was done
by random selection, and all clinical and
laboratory assays were performed under
double-blind, placebo-controlled conditions.
Viral detection and identification were done
in LLC-MK2 tissue culture tubes with
cytopathic effect as an endpoint. Antibody
assays were done by a "standard" hemag-
glutination-inhibition test and by a micro-
titer neutralization test using LLC-MI<2
cells.
Results and Discussions
Six separate studies were conducted
using a total of 189 subjects. Forty
subjects participated in Study 1, in which
tap water at the isolation site was used as
the vehicle for inoculation. About 45
minutes prior to ingestion by the subjects,
the virus doses were suspended in tap
water, having a free residual chlorine of
0.8 mg/l. Subsequent tests indicated that
100 pfu of Echovirus-1 2 suspendeo ,.i
this tap water was reduced to a non-
detectable level within 45 minutes when
assayed in LLC Ml<2 cell culture. At the
dosage levels administered in experiment
1, subsequent feeding experiments in-
dicated that 18 of the subjects administered
virus would likely have become infected if
the inoculum had been fully viable. How-
ever, none of the 30 subjects who ingested
virus suspended in chlorinated tap water
became ill or yielded evidence of infection.
Chilled distilled Talawanda water* was
used as a vehicle in the remaining five
studies.
Study 2 was a range-finding study,
which did not include placebo controls.
Viral doses ranged from 10-10,000 pfu.
Dosages in subsequent studies ranged
from 10-300 pfu.
The following results are based on the
149 subjects who participated in studies
2-6. There was no evidence of infection or
illness amortg the placebo subjects. No
illness occurred among any virus-inocu-
lated subjects However, a "dose-response"
curve for infection was established. Evi-
dence for infection was based primarily on
isolation of Echovirus-12 from fecal swabs.
Only four subjects experienced significant
antibody responses in the absence of
intestinal viral shedding. Likewise, only
five subjects were found to shed virus in
the pharynx who were not found to be
shedding virus in the intestinal tract and in
these, virus was found on only one day in
each case. The majority of intestinal viral
shedding occurred during the first week
after administration of viral inocula and
persisted for several weeks in some sub-
jects, regardless of the dose.
Using the definition of infection as re-
covery of virus from fecal specimens
and/or a significant serological response,
the infection rates were as follows:
Dose(pfu) No. subjects No. infected-96
0
10
30
100
300
1,000
1 0,000
34
50
20
26
12
4
3
0
15
9
19
12
2
2
( 0)
( 30)
( 45)
( 73)
(100)
( 50)
( 67)
Using a probit analysis based on the
values presented in the above data for four
dosages (10, 30, 100, 300 pfu) it can be
inferred, within 95% fiducial limits, that 1 -
2 pfu would infect 1 % of a susceptible
population.
Because of the implications of the re-
sults found in this study in regard to public
health practices and policies, the following
points must be considered carefully:
1. The virus used in the study was
"safe" and not associated with seri-
ous natural disease. Additional stud-
ies using other enteric viruses should
be conducted.
* Mention of trade names or commercial products does
not constitute endorsement or recommendation for
use by the U S Environmental Protection Agency.
2. An "end point" (a dose which did not
cause infection) was not determined.
Accurate delivery of a dose less than
10 pfu is not statistically sound un-
less very large numbers of volun-
teers are studied. Therefore, statisti-
cal inference was required to deter-
mine a minimal infectious dose.
3. "Susceptibility" was defined as no
detectable serum antibody at a 1:5
dilution of serum by HI and/or dilu-
tion by microtiter neutralization. No
effort was made to determine pre-
existing intestinal antibody.
4. Only one cell culture assay system
was used to titer the viral inocula.
Conclusions
1. No clinical illness occurred following
administration of 10-10,000 pfu of
Echovirus-1 2.
2. Infection was detected in 30% of the
subjects giveq 10 pfu indicating that
the minimum infectious dnse may be
considerably lower.
3. Using statistical analysis and extrap-
olation, it was determined that 1-2 pfu
may be infectious for 1 % of the popu-
lation.
4. Intestinal shedding at doses as low as
10 pfu may persist for at least 1 9
days. At higher doses, the persistence
may be longer.
5. Secondary spread was not detected
among susceptible adult contacts.
6. Virus ingested in drinking water may
sometimes be recovered from the
pharynx
7. Consistent significant serum antibody
responses probably require a greater
dose (< 1 0,000 pfu) or a different
route of administration.
8. Chlorine-inactivated virus ingested at
a concentration up to 100 pfu did not
yield a detectable infectious or im-
mune response.
Recommendations
:. Similar studies should be conductec
using different viral inocula that mighi
have greater medical significance (yet
still be safe), e.g., other enteroviruses
rotaviruses.
2. In order to determine the effect o
prior infection on susceptibility to lov
viral doses, the Echovirus-12 stud'
should be extended to include sub
jects with naturally-acquired serun
antibody; those subjects from the cur
rent study who received virus am
developed serum antibodies, and alsi
those who received and shed virus bu
did not develop antibodies.
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3. Consideration should be given to an
investigation of the role of multiple
viral ingestions (i.e., more than one
viral type at the same time; the same
virus several times) and different
routes of inoculation, e.g., aerosol.
4. Future investigations should include
the collection of pharyngeal washings
to further determine the incidence of
pharyngeal shedding subsequent to
viral ingestion in water.
5. Attempts should be made to deter-
mine viral particle content in relation
to plaque-forming units in infective
dose studies.
6. Evaluation should be made of other
cell lines and assay procedures to deter-
mine infectivity titers for "challenge"
viruses. If more sensitive cell lines are
found, additional studies in human
subjects should be performed to di-
rectly measure viral infectivity at lower
doses than used in the present study.
7. Viral shedding should be more ac-
curately quantitated to determine the
relationship between dose and viral
replication.
8. The relationship between viral infec-
tion and local (intestinal) antibody pro-
duction should be determined, as well
as the length of time such antibody is
produced.
Gilbert M. Schiff, Gerda Stefanovic. Betsy Young, and Julia Pennekamp are with
The Christ Hospital Institute of Medical Research, Cincinnati, OH 45219.
Elmer Akin is the EPA Project Officer (see below).
The complete report, entitled "Determination of Minimal Infectious Dose of an
Enterovirus in Drinking Water," (Order No. PB83-191 015; Cost: $8.50, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
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
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Environmental Protection
Agency
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Information
Cincinnati OH 45268
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