&EPA
United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/M-89/028 Jan. 1990
ENVIRONMENTAL
RESEARCH BRIEF
Electron Microscopic Examination of Gf'ard/a Cultures for Viruses
Fred P. Williams, Jr.*
Abstract
Giardia lamblia is an important waterborne pathogen in
the United States. Laboratory cultures of G. lamblia are
maintained by USEPA researchers who conduct
disinfection studies with this pathogen or who develop
increasingly effective methods to detect it in the
environment. Some G. lamblia cultures maintained in
other laboratories have recently been shown to be
infected with a virus. Researchers are concerned that
infected cultures may react differently in laboratory
studies. In this study, electron microscopy (EM) was used
to examine USEPA cultures of G. lamblia for virus. Virus
was demonstrated in one of four G. lamblia strains
examined by this method. The infected strain will be used
to determine the consequences of viral infection.
Introduction
Recently, investigators preparing a G. lamblia cDNA
library discovered a virus infecting the protozoan (1)- Tne
identified virus is roughly spherical, 33-37 nm in diameter,
and has a dsRNA genome 7 kb in size. The G. lamblia
virus (GLV) bands in a CsCI gradient at a buoyant density
"Environmental Monitoring Systems Laboratory, U. S Environmental
Protection Agency, Cincinnati, OH 45268
of 1.368 g/ml. Analysis of the purified virus has revealed a
single major protein species of 100,000 molecular wt (2).
GLV has been identified in G. lamblia cultures obtained
from a variety of sources and has been found in isolates
of both human and animal origin (3). Not all G. lamblia
cultures are infected. Virus-free cultures have been
demonstrated by various investigators (3,4). While some
virus-free cultures are susceptible to infection with GLV,
others appear to be resistant. GLV does not cause cell
lysis, and infected cultures are not readily identifiable
without special methods to detect the virus. The
significance of GLV infection is not presently clear.
To determine if G. lamblia strains cultured and studied in
EMSL-Cincinnati parasitology laboratories are infected
with GLV or other viruses, preparations of these cultures
were examined for the presence of virus particles using
EM.
Materials and Methods
G. lamblia cultures. Axenic trophozoite cultures of four
different G. lamblia strains were examined. These strains
included Human-1-Portland (H-1-P), CDC:0284:1, ATCC
30957, and ATCC 50137. The specimens for EM exam-
ination were obtained as trophozoite suspensions in 1X
Hanks' Balanced Salt Solution (HBSS). The suspensions
were placed through three freeze-thaw cycles before
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examination. In one case (H-1-P), the crude freeze-thaw
preparation was further processed to extract virus and
remove cellular debris (after removing an aliquot of the
crude preparation for EM examination). Virus particles
which may have been entrapped in H-1-P cellular material
were eluted with 3% beef extract and extracted with
trichlorotrifluoroethane. After low-speed centrifugations to
remove unwanted material (170 X g for 15 min and 3500 X
g for 30 min), the possible virus component was pelleted
by high-speed centrifugation at 110,000 X g for 2 hr. The
resulting pellet was resuspended with 0.5 ml of HBSS and
this suspension examined by EM.
Electron microscopy. Direct negative-stain examination of
the four crude freeze-thaw preparations and single purified
preparation was carried out as previously described (5).
Briefly, a drop of one of the preparations was applied to an
EM grid with carbon support film. After 1 min, excess
material was removed from the grid with filter paper. The
grid was rinsed with 1 or 2 drops of distilled water and
then stained with 2% phosophotungstic acid, pH 7. Excess
stain was removed with the filter paper. After drying, the
grid was examined at 80 kV with a JEOL JEM-100CX
electron microscope.
To optimize viral adsorption and distribution, some grids
were pretreated with 0.1% poly-L-lysine (6) before
specimen application. Each preparation was examined
using both poly-L-lysine pretreated and untreated grids.
Cyiopathic effect (CPE) in animal cell culture. Two 0.25 ml
aliquots of the H-1-P purified preparation were used to
inoculate two 6 oz culture bottles containing confluent
monolayers of buffalo green monkey (BGM) kidney cells.
The inoculated cell monolayers were examined daily for
the development of CPE as compared to uninoculated
control monolayers.
Plaque formation in bacterial culture. Serial dilutions of the
H-1-P purified preparation were made and 0.5 ml aliquots
of each dilution were assayed for plaque forming units
according to a procedure described elsewhere (7). The
plaque assay was performed in triplicate using three
different E. coli strains as the host bacterium. The strains
included E. coli C (ATCC 13706), E. coli C-3000 (ATCC
15597), and E. coli A-19 (An Hfr strain obtained from R. L.
Ward, Gamble Institute for Medical Research, Cincinnati,
OH).
Results
Results of the EM examination of the G. lamblia cultures
are presented in Table 1. Virus particles were visualized in
both the crude freeze-thaw preparation and the purified
preparation of strain H-1-P (Figure 1; A, B, and C). The
virus particles were visualized using both untreated EM
grids and grids pretreated with poly-L-lysine. The particles
appeared to be more abundant in the crude preparation
than in the purified preparation. Massive aggregates of
virus particles were observed in the crude preparation
(Figure 2 and Figure 3). These massive aggregates were
not apparent in the purified preparation. Such aggregates
were likely removed or dispersed during the further
processing of the crude preparation. No virus particles,
similar or of other type, were detected in the crude
preparations of the other three G. lamblia strains.
The virus particles appeared generally spherical with only
slight indication of an icosahedral nature. Most of the
Table 1. Results of EM Examination of Giardia Cultures
for Viruses
Virus Particles Visualized
G. lamblia
Strain
H-1-P
H-1-P
CDC:0284:1
ATCC 30957
ATCC 50137
Type of
Preparation
Crude
Purified
Crude
Crude
Crude
Untreated Pretreated*
Grids Grids
+ +
+ +
-
-
-
*EM grids pretreated with 0.1% poly-L-lysme before sample
application.
particles were observed to be penetrated by the negative
stain. The mean diameter of 91 particles ±SD was 38 ± 2
nm. The particles revealed no capsomeric detail, and no
distinctive structural features were evident.
CPE was not observed in the two BGM cultures that were
inoculated with the virus-particle-containing H-1-P prep-
aration. Inoculated monolayers appeared intact and iden-
tical to uninoculated control monolayers through day 21
post inoculation.
Plaques were not detected in the bacterial cultures of E.
coli C, A-19, and C-3000 that were inoculated with the H-1-
P preparation.
Discussion
The virus particles visualized in the H-1-P culture
preparations appear to represent the same virus (GLV) that
was detected by Wang and Wang (1986). Although those
investigators originally reported particles 33 nm in
diameter (1), they later reported a diameter of 37 nm for
GLV (2). This latter figure corresponds well to the mean
diameter of 38 nm found for the particles observed in the
present investigation. Additionally, published electron
micrographs of GLV reveal particles readily penetrated by
negative stain. This was also observed in the present
investigation. It should be noted that these stain-penetrated
particles should not be considered defective in lacking
their nucleic acid component. Similar yeast virus particles,
which also appear to be readily stain-penetrated, have
been shown to have their nucleic acid present (8,9).
Cells (BGM) routinely used to detect viruses of the human
gastrointestinal tract did not develop CPE after being
inoculated with the H-1-P particles. Similar negative
findings were reported by Wang et al. (1988) using two
human intestinal cell lines. The particles also caused no
lysis of bacterial cultures using E. coli strains known to be
susceptible to small isometric bacterial viruses such as
MS2and4>X174.
Mention has been made of a morphological resemblance
between GLV and minirotavirus particles (1). Minirotavirus
particles have been described as being 32 nm in diameter.
They exhibit an "irregular margin, at times resembling a
palisade of very small capsomeres" (11). Such char-
acteristics are consistent with the "small round structured
virus" (SRSV) classification proposed by Caul and
Appleton (1982). In the present investigation, the visualized
particles exhibited no such characteristics (and SRSVs
have often been observed in this laboratory using the
same negative-stain procedures). These particles can best
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Figure 1. (A and B) Virus particles observed in the negatively stained crude freeze-thaw preparation of G. lamblia H-1-P.
(C) Membrane-associated particles, some in hexagonal array, in the purified H-1-P preparation. Bar = 100 nm for
A, B, and C.
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F!g.3
Figures 2 and 3. Massive aggregates of virus particles observed in the crude preparation of G. lamblia H-1-P. Bar = 0.5 p.
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be described as being "featureless" (12) with regard to
morphological appearance.
Acknowledgment
The G. lamblia propagation and preparative work was
performed by Joseph M. Bifulco and Dr. Frank W.
Schaefer, III, Parasitology and Immunology Branch, MRD,
EMSL-Cincinnati. The bacterial culture plaque assay of the
H-1-P preparation was performed by Alvin G. Jose,
Virology Branch, MRD, EMSL-Cincinnati.
References
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