APPLIED MICROBIOLOGY, Oct. 1974, p. 717-719
Copyright 0 1974
American Society for Microbiology
Vol. 28, No. 4
Printed in U.S.A.
Concentration of Enteric Viruses from Water with Lettuce
J. KONOWALCHUK, J. I. SPEIRS, R. D. PONTEFRACT, AND G. BERGERON
Food Research Laboratories, Health Protection Branch, Health and Welfare Canada, Ottawa, KIA OL2,
Received for publication 22 July 1974
A method for recovering enteroviruses, adenovirus, and reovirus from water
with lettuce extract is described. Lettuce extract at pH 8.5 was added to the
sample and the pH was reduced stepwise with hydrochloric acid to 4.0 to 4.5. The
flocculent lettuce-extract particles, and adsorbed virus, were readily removed
from solution by low-speed centrifugation. Electron microscopy suggests that,
under conditions suitable for adsorption, virus particles are coated with the
A number of methods for the recovery of
virus from water have been described recently
(1, 4-7). Virus adsorption followed by elution
has been the principal approach for virus con-
centration. In an earlier report (2), we described
a method for enterovirus recovery using lettuce
floc. The work has been extended to include two
other enteric viruses, reovirus 1 and adenovirus
Lettuce extract was prepared as described
previously (2). The extract is a clear amber-col-
ored colloidal suspension at pH 5.5 or above. A
floc forms below pH 5.5 and is readily sedi-
mented by low-speed centrifugation at pH 4.0 to
4.5. The dry weight of the floc varied from 1.5 to
3.0 mg/ml depending on the batch. Approxi-
mately 50% of the dry weight was protein, as
determined by the method of Lowry et al. (3).
Adsorption to lettuce extract of coxsack-
ievirus types B4 and B5, echovirus type 7,
poliovirus type 1 (Sabin), reovirus type 1, and
adenovirus type 7a was examined as follows.
Virus concentrations of 100 or 1,000 plaque-
phate-buffered saline were added individually
to samples containing 10 to 1,000 ml of distilled
water. Final virus concentrations varied from
0.1 to 100 PFU per ml of sample. A similar
inoculum was added to duplicate samples of 2.5
ml of growth medium to serve as virus controls.
A 10% volume of lettuce extract was added to
the water samples at pH 5.0, 6.0, 7.0, 8.0, or 8.5.
Samples were adjusted to pH 4.0 to 4.5 in 0.5 to
1 log steps by dropwise addition of HC1. After
centrifugation at 1,000 x g for 10 min, the
pellets were dissolved by adding NaOH; 0.05 ml
of 1 N NaOH dissolved the 0.4- to 0.8-ml pellet
obtained from a 200-ml sample. Water samples
larger than 200 ml were divided, centrifuged,
and finally recombined after dissolution of the
pellets. The sample was diluted to 2.5 ml with
strength medium and then was assayed on a
single monolayer of cultured cells as described
previously (2). HEp-2 cells were used for the
coxsackievirus and poliovirus, Vero cells were
used for echovirus, and primary African green
monkey kidney cells were used for reovirus and
adenovirus. Enterovirus plaques were read after
3 days, adenovirus and reovirus after 8 days.
Results of preliminary experiments indicated
that virus in the dissolved pellet alone or with
added 10% serum would not adsorb to cell
Earle, or saline with or without 10% serum,
however, permitted infection of monolayers.
pH OF LETTUCE EXTRACT
FIG. 1. Effect of pH on virus recovery from water
samples treated with lettuce extract. Results of three
different experiments were averaged.
FIG. 2. Reovirus treated with lettuce extract. (A) pH 6.0, not coated; (B) pH 8.5, coated. x 187,000.
FIG. 3. Adenovirus treated with lettuce extract. (A) pH 4.5, not coated; and (B) pH 6.0, coated. x 193,000.
VOL. 28, 1974
Coxsackievirus B4, B5, echovirus 7, polio-
concentrated from water with colloidal lettuce
extract at pH 6.0 or higher. Flocculent extract
at pH 5.0 was less efficient in concentrating
quired a pH of 8.0 for effective concentration
Quantitative recovery of all viruses tested was
accomplished by adding lettuce extract at pH
8.5 to the water sample followed by the drop-
wise addition of HCl in 0.5 to 1 log steps to pH
4.0 to 4.5. Virus inputs varying from 0.1 to 100
PFU per ml of sample were concentrated from
water volumes of 10 to 1,000 ml. Concentrates
from volumes as great as 500 ml could be
assayed in one plastic dish without apparent
toxicity to the cell monolayer. Less than 1% of
the virus input remained in the supernatant
fluid as unadsorbed virus. Mixed as well as
unmixed populations of reovirus and adenovirus
For electron microscopy, viral suspensions
were diluted with 2% potassium phosphotung-
state at pH 6.8 and spread on pure carbon or
grids. The carbon surface was rendered hydro-
philic by a brief treatment (ca. 40 s) of exposure
to ionized air in a Plasmod unit (Tegal Corp.,
Richmond, Calif.). The negatively stained vi-
ruses were then examined at a magnification of
60,000 to 100,000 in a Siemens Elmiscop 101.
Electron photomicrographs showed that virus
was coated with colloidal particles or aggregates
1, and adenovirus 7a were efficiently
of them at optimal pH levels. Capsomers of
reovirus were sharply defined at pH 6.0, but
lettuce-extract colloid (Fig. 2). Adenovirus was
well defined at pH 4.5 but hazy at pH 6.0 (Fig.
3). Controls of reovirus and adenovirus without
lettuce extract at similar pH measurements
were all sharply defined. Colloidal particles
were a few nanometers in diameter; some aggre-
gates of the particles were as large as 100 nm.
By following the procedure outlined above,
enteroviruses, adenoviruses, and reoviruses are
efficiently removed and concentrated
at pH 8.5 due to adsorbed
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Metcalf. 1972. Virus in water. II. Evaluation of mem-
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2. Konowalchuk, J., and J.
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I. Speirs. 1973. Enterovirus