Comparative Replication of HSV-1 in BALB/c
and C57BL/6 Mouse Embryo Fibroblasts In Vitro
5. Z. AbghQri,*'t't R. Doyle Srulring,* S. M. Nigida, Jr.,f Donald N. Downer,^: ond Andre J. Nohmiost
Herpes simplex virus type 1 (HSV)-host cell interactions were studied in fibroblasts from inbred mice
by measuring virus replication, virus adsorption, infectious center formation, and single-cell virus production. BALB/c mouse embryo fibroblasts (MEF) produced more intracellular and extracellular virus
than C57BL/6 MEF, with differences in virus production first appearing at 16 hr after inoculation.
Virus yield in C57BL/6 cells peaked earlier (16 hr) and at a lower level than in BALB/c cells (20 hr).
These results were explained by a difference in single-cell virus replication, rather than less efficient
adsorption or the presence of cells that could not be infected. Host-related variation in the ability of
infected cells to support HSV replication may account, in part, for differences in the severity of HSV
ocular disease. Invest Ophthalmol Vis Sci 27:909-914, 1986
Previous studies have shown that inbred BALB/c
mice are more susceptible than C57BL/6 mice to
herpes simplex virus type 1 (HSV) stromal keratitis.1'2
It is generally believed that the host immune response
to viral antigen(s), rather than the lysis of cells by replicating virus, plays a major role in tissue destruction
in some forms of HSV stromal keratitis.3"6 However,
the quantity of infectious virus recovered from tissues
of inbred mice relatively susceptible to stromal keratitis
is greater than that recovered from relatively resistant
mice, suggesting that a difference in the ability of virus
to replicate might help explain individual differences
in susceptibility.7 This hypothesis is supported by our
finding of a difference in the ability of cell monolayers
from inbred mice to allow the replication of HSV in
vitro.2 Differences in the ability of cell monolayers to
produce virus may be due to differences in the ability
of cells to adsorb virus, differences in the proportion
of infectible cells present in the monolayer, or differences in virus production by each infected cell. We
have extended our previous investigations by examining the mechanism of virus-cell interaction that accounts for more efficient replication of HSV in cells
from susceptible mice than in cells from resistant mice.
Materials and Methods
Cell culture medium: Growth medium (GM) was
Eagle's minimal essential medium (MEM) containing
5% fetal calf serum, 5% newborn calf serum and antibiotics as described previously.7 Maintenance medium (MM) was MEM containing 1% fetal calf serum,
1% newborn calf serum and antibiotics.
Continuous cell lines: Vero and HEp-2 cell lines were
grown and maintained as previously described.7
Primary mouse kidney cells: Kidneys from 6- to 8wk-old BALB/c and C57BL/6 mice were removed,
washed, and minced. Animals were handled in compliance with the ARVO Resolution on the Use of Animals in Research. Tissue fragments were rinsed in calcium and magnesium-free phosphate-buffered saline
(0.01 M, pH 7.6) and suspended in 0.25% trypsin in
Hanks' salt solution. After slow stirring at 4°C overnight, cell suspensions were filtered through sterile
mesh cloth, resuspended in GM (1.0 X 106 cells/ml)
and placed in 75 cm2 tissue culture flasks. Medium
was changed every 3 to 4 days. Confluence was reached
3 to 4 days after seeding.
Primary mouse embryo fibroblasts (MEF): MEF
were cultured as previously described2 and used at the
second to fifth passage.
Mouse keratocytes: Keratocytes were cultured as
previously described2 and used at the third passage.
Virus: Isolate 4, the herpes simplex virus type 1 used
in these studies, was obtained from a patient with herpetic keratitis and produces stromal keratitis in mice.2
Virus stocks were prepared in HEp-2 cells and stored
at —70°C. The titer was determined in vero cells. For
use in experiments, stock virus was diluted in PBS
From The Emory School of Medicine, Departments of Ophthalmology* and Pediatricst and from Mississippi State University, Department of Biological Science.:):
Supported by NIH grants EY-05097, AI-19554, and grants from
Research to Prevent Blindness, Inc., the Georgia Lions Eye Bank,
Inc., and the Seymour R. Marco Foundation.
Submitted for publication: July 10, 1985.
Reprint requests: R. Doyle Stulting, MD, PhD, Department of
Ophthalmology, 1327 Clifton Road, N.E., Atlanta, GA 30322.
909
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910
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1986
(0.01 M, pH 7.3), containing 1% fetal calf serum and
0.1% glucose (PBS-A).
Kinetics of HSV replication in vitro: Confluent
monolayers (0.7-1.0 X 106 cells/25 cm 2 flask) were infected with HSV at a multiplicity of five plaque-forming
units (PFU)/cell. Virus was allowed to adsorb to the
cells for one hour at 4°C. The inocula were then aspirated, the cell monolayers were washed twice with
PBS, MM (3.0 ml) was added to each flask, and the
flasks were incubated at 37°C. At intervals, extracellular and intracellular infectious virus were separated
by the following procedure. To recover extracellular
virus, 1.0 ml of culture supernatant fluid was removed
from each flask, centrifuged (500 X g, 10 min), and
the supernatant fluid was frozen at -70°C. To recover
intracellular virus, the infected cells were scraped from
the surface of the flask, added to the cells remaining
in the centrifuge tube, and again centrifuged. The supernatant fluid was discarded. The cells were washed
twice in PBS, resuspended in MM (3.0 ml), freezethawed, sonicated, centrifuged (500 X g, 10 min), and
supernatant fluid was frozen. All samples from each
experiment were thawed and assayed on vero cells at
the same time.
Relative sensitivity of cells to HSV infection: Confluent cell monolayers were inoculated with the same
stock solution of HSV and plaques were counted. The
diameter of 20 individual plaques was determined microscopically using a stage micrometer slide graduated
in 0.1-mm increments.
Adsorption assay: Confluent monolayers of mouse
embryo fibroblasts were trypsinized and suspended in
GM. Cell suspensions were centrifuged (500 X g, 10
min) and the cell pellets were washed twice with PBS.
Cells were then counted and adjusted to a concentration of 1.0 X 106/ml. Cell suspensions (1.0 ml) were
transferred to small tubes, centrifuged (500 X g, 10
min), and the supernatant fluids were discarded. Cell
pellets were resuspended in 1.0 ml of PBS-A containing
HSV (1.0 X 104 PFU/ml) and incubated at 4°C with
occasional agitation. Virus incubated in tubes without
cells was used to determine zero adsorption. After 1
hr, the tubes were centrifuged (500 X g, 10 min), and
the amount of virus present in the supernatant fluids
was determined on vero cells by plaque assay.8 The
percent of virus adsorbed was calculated according to
the following formula:
Adsorption (%)
Titer of virus in tubes without cells
— Titer of virus in tubes with cells
X 100.
Titer of virus in tubes without cells
Infectious center assay: MEF monolayers were infected with HSV at a multiplicity of five. After incu-
Vol. 27
bation (37°C, 16 hr), cells were scraped into the medium and a single cell suspension was obtained by repeated pipetting. One hundred cells in 1.0 ml were
inoculated onto a vero cell monolayer and incubated
at room temperature for 5 hr. One ml of MM containing 0.2% human gamma globulin (Cutter Biological, Inc.; Berkley, CA) was then added to each well
and the incubation was continued for 48-72 hr (37°C,
5% CO2). The number of infectious centers per 100
cells was determined after monolayers were stained
with crystal violet (0.5% in 20% ethanol).
Single cell virus production: Confluent MEF monolayers were infected with HSV as above. Infected
monolayers were incubated for 7 hr (37°C, 5% CO2),
then the cells were scraped into the medium. After
centrifugation (500 X g, 10 min), the cells were rinsed
twice with PBS and resuspended in MM at a concentration of 2 cells/ml. Aliquots of 0.2 ml were transferred
to micro test tubes so that single cells would be expected
to be found in two out of five tubes. Tubes were incubated for 16 hr (37°C, 5% CO2). The virus present
in each tube was quantified by plaque assay using inocula of 0.2 ml.8
Results
Replication of HSV in MEF monolayers: Intracellular and extracellular virus production in BALB/c,
C57BL/6, and HEp-2 cell monolayers was measured
at 24 hr post-inoculation (PI). The titer of intracellular
virus produced in BALB/c MEF varied between 8.7
and 19 X 106 PFU/10 6 cells; the titer of intracellular
virus produced in C57BL/6 MEF was 9- to 12-fold less
(0.98 to 1.6 X 106 PFU/10 6 cells) (Table 1). Quantification of extracellular virus gave similar results, with
8- to 13-fold more virus produced in BALB/c MEF
than in C57BL/6 MEF. In all experiments, both intracellular and extracellular virus replication were most
efficient in HEp-2 cells.
Kinetics of replication of HSV in MEF cells: Differences in total intracellular virus present after 24 hr of
incubation were not due solely to differences in the
kinetics of virus replication, since virus production was
greater in BALB/c than C57BL/6 monolayers at all
times after eclipse (Fig. 1). Peak intracellular virus production was more than tenfold greater in BALB/c MEF
monolayers than in C57BL/6 MEF monolayers, although peak production occurred at different times (20
hr for BALB/c vs 16 hr for C57BL/6). Similar results
were obtained for extracellular virus, with differences
in virus production becoming evident sooner and peak
production occurring at 24 hr for BALB/c and 16 hr
for C57BL/6 (Fig. 2). Both intracellular and extracellular virus production in MEF monolayers was less
than that in the continuous human cell line HEp-2
(Figs. 1 and 2).
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911
REPLICATION OF HSV-1 IN MOUSE FIDRODLASTS/Abghori er ol.
No. 6
Table 1. Production of herpes simplex virus by infected mouse embryo fibroblast
monolayers at 24 hr postinoculation
Virus recovered
Intracellular
Extracellular
Experiment
HEp-2
BALB/c
C57BL/6
P-value*
HEp-2
BALB/c
C57BL/6
P-value
1
2
3
8.20 X 10 7 t
9.00 X 107
1.04 X 108
8.70 X 10*
1.30 X 107
1.91 X 107
9.83 X 10s
1.31 X 10*
1.57 X 10*
<0.01
<0.05
<0.05
9.2 X 10*
1.14 X 107
3.19 X 10*
2.77 X 10*
9.90 X 105
2.69 X 10*
2.13 X 10s
1.29 X 10s
2.13 X 10s
<0.05
<0.02
<0.01
* BALB/c vs C57BL/6 (Student's t-test). For all three experiments combined,
BALB/c monolayers produced more virus than C57BL/6 monolayers (P
< 0.001).
Sensitivity of cells to HSV infection: Keratocytes,
kidney cells, and MEF from BALB/c and C57BL/6
mice were inoculated with the same stock virus suspension. Eight- to nine-fold more plaques were formed
Intracellular Virus
f P F U p e r 10* cells.
in BALB/c monolayers, compared to C57BL/6 monolayers for all cell types examined (Fig. 3). The average
plaque diameters in BALB/c and C57BL/6 MEF were
not statistically different (0.82 mm and 0.85 mm respectively; P > 0.8, Student's t-test).
Adsorption ofHSVbyMEF: Host-related differences
in HSV replication may be due to differences in virus
Extracellular Virus
7
10 -
• HEp-2
• BALB/c
A C57BL/6
• HEp-2
• BALB/c
C57BL/6
i
i
r
i
8
12
16
20
24
28
8
Hours after Inoculation
Fig. 1. Quantification of intracellular HSV production in cultured
inbred mouse cells. BALB/c and C57BL/6 MEF and HEp-2 monolayers were infected with HSV at a multiplicity of five. Intracellular
virus production was measured at 0-28 hr post-inoculation. Differences in intracellular virus production by BALB/c and C57BL/6 MEF
were statistically significant (Wilcoxon matched pairs signed rank
test; P < 0.01).
12
16
20
Hours after Inoculation
Fig. 2. Quantification of extracellular HSV production in cultured
inbred mouse cells. BALB/c and C57BL/6 MEF and HEp-2 monolayers were infected with HSV at a multiplicity of five. Extracellular
virus production was measured at 0-28 hr post-inoculation. Differences in extracellular virus production by BALB/c and C57BL/6
MEF were statistically significant (Wilcoxon matched pairs signed
rank test; P < 0.05).
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912
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1986
Plaque Formation by HSV
in Cultured Inbred Mouse Cells
108n
Vol. 27
Table 3. Infectious center assay for BALB/c and
C57BL/6 mouse embryo fibroblasts
Infectious centers/100 infected
MEF from
Experiment
number
BALB/c
C57BL/6
HEp-2
1
2
3
93
102
96
92
100
94
92
99
98
10-
e
I
!•
u_
Q.
I
iU HEp-2
EDvero
BALB/c
* Results are not significantly different for BALB/c and C57BL/6 (Student's
t-test; P > 0.80).
I
10 6 "
Reference
Cell Lines
Cornea Kidney Embryo
Fig. 3. Plaque formation by HSV in cultured inbred mouse cells.
Confluent monolayers of HEp-2 cells, BALB/c MEF, and C57BL/6
MEF were infected with HSV and plaques enumerated. More plaques
were produced in BALB/c cells than in C57BL/6 cells (Student's ttest; P < 0.001 for all tissues).
adsorption or differences in post-adsorption events. No
significant differences in the ability of BALB/c and
C57BL/6 MEF to adsorb virus were found (Table 2).
Infectious center assay: Since the MEF were used
during early passages (two through five), these cultures
were likely to contain diverse cell types. Increased virus
production by BALB/c MEF compared to C57BL/6
MEF could therefore have resulted from infection of
only a fraction of the cells present, with a higher percentage of infectable cells in the BALB/c MEF monolayers than in the C57BL/6 MEF monolayers. To examine this possibility, infectious center assays were
performed. Three separate experiments showed no difTable 2. Adsorption of herpes simplex virus by
BALB/c and C57BL/6 mouse embryo fibroblasts
ference in the percentage of BALB/c and C57BL/6 cells
that were infected at a multiplicity of five (Table 3).
Replication in isolated cells: Cultures of BALB/c
MEF produce more HSV after infection than do cultures of C57BL/6 MEF (Table 1). Since the cell cultures
have a similar ability to adsorb virus (Table 2), and
essentially all cells in a culture can be infected (Table
3), then each BALB/c cell should produce more virus
than each C57BL/6 cell. This hypothesis is confirmed
by the data presented in Table 4, which show that single
infected BALB/c MEF produce 4- to 6-fold more virus
than single infected C57BL/6 MEF.
Discussion
The severity of herpetic ocular disease in inbred mice
is dependent on both the host and virus strain. Following topical ocular inoculation with HSV, more
BALB/c mice develop stromal keratitis than do similarly inoculated C57BL/6 mice.1'2 More infectious virus
can be recovered from the inoculated eye, the ipsilateral
trigeminal nerve, and the ipsilateral trigeminal ganglion
of susceptible BALB/c mice than from corresponding
tissues of resistant C57BL/6 mice.7
Lopez reported that replication in vitro of HSV
in embryo fibroblasts from susceptible (A/J and
BALB/c) strains of mice was not different from replication in the resistant (C57BL/6) strain studied.9 Cook
and Stevens found no significant difference in the abil-
Virus liter
Experiment
1
2
Cell type
Unadsorbed
Adsorbed
Percent
adsorbed
None
HEp-2
BALB/c
C57BL/6
11.00*
4.22
3.00
3.37
_
6.78
8.00
7.63
_
62
73
69
None
HEp-2
BALB/c
C57BL/6
5.20
1.28
1.70
1.60
3.90
3.50
3.60
75
67
69
* PFU X 102. There is no significant difference in adsorption of HSV by
BALB/c and C57BL/6 fibroblasts P> 0.10; (Student's t-test).
Table 4. Herpes simplex virus production by single
isolated cells infected in vitro
Mouse strain
Experiment
HEp-2
BALB/c
C57BL/6
P-value
(BALB/c vs
C57BL/6)
1
2
3
8.8* (12/16)f
4.6 (13/24)
5.2 (6/15)
6.8(5/17)
6.7(13/24)
4.7(6/15)
1.2(5/16)
1.5(13/24)
1.2(6/15)
0.004 $
0.002
0.001
* Mean number of plaques per positive culture.
t Positive cultures per number of cultures seeded.
t Mann-Whitney U test.
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No. 6
REPLICATION OF HSV-1 IN MOUSE FIBROBLASTS/Abghari er ol.
ity of spinal ganglia from susceptible and resistant
inbred mice to support HSV replication in vitro.10
These authors concluded that the intrinsic ability of
structural cells to support virus replication plays no
role in the development of disease. In contrast, we and
others have shown that embryo fibroblasts and keratocytes from susceptible strains replicate more virus
during a 24-hr period than do fibroblasts and keratocytes from resistant strains. 2 " The data presented (Table 1) corroborate these results.
Further experiments were performed to determine
if this difference in virus produced during 24 hr was
due solely to differences in the kinetics of virus replication with peak production occurring at different
times and the total amount of virus recovered being
influenced by virus inactivation during the incubation
period. The time of peak virus production was indeed
different for the two strains (Figs. 1 and 2), but the
amount of virus recovered from BALB/c MEF was
greater than that recovered from C57BL/6 MEF at all
times. These findings confirm genetically determined
differences in the ability of host cells isolated from the
influence of the immune system to support HSV replication. The data are in agreement with the results of
Collier et al, who found host strain-related differences
in HSV replication using DBA/2 and C57BL/6 embryo
fibroblasts.11
The difference in the in vivo susceptibility of different
mouse strains to HSV parallels the in vitro replication
of HSV in structural cells from these same strains. This
finding suggests that variation in the ability of structural
cells to restrict HSV replication may explain, at least
in part, differences in host susceptibility to HSV ocular
disease. Our in vitro assays were designed to elucidate
further the mechanism by which cells from resistant
mouse strains interact with HSV in vitro.
Eight to nine times more plaques are produced by
HSV in BALB/c MEF than in C57BL/6 MEF (Fig. 3).
Not only do infected monolayers from susceptible
BALB/c mice produce more virus, but also more foci
of infection are established under similar conditions of
inoculation. Thus, quantitative differences in virus recovery following topical ocular inoculation in vivo7
may be due not only to differences in the ability of
cells to support virus replication, but also to differences
in the number of foci of infection that are established.
Adsorption assays revealed that HSV adsorbs to
C57BL/6 MEF as efficiently as to BALB/c MEF. This
result suggests that restricted HSV replication in
C57BL/6 MEF is not related to cell surface antigen
heterogeneity or to variation in the ability of virus to
bind to cell receptors.
Since early passage monolayers were used in these
experiments, the cell populations in each monolayer
913
could be heterogenous, with some cells being incapable
of becoming infected with HSV. Therefore, differences
in virus yield could be related to variation in the number of infectible cells present in the monolayers. The
infectious center assay (Table 3) showed that virtually
all cells of both strains can be infected at a multiplicity
of five, indicating that the presence of uninfectable cells
does not account for host strain-related differences in
virus production.
Since the amount of HSV adsorbed by BALB/c and
C57BL/6 MEF is the same and noninfectible cells are
not present, isolated BALB/c MEF should support
replication of HSV better than isolated C57BL/6 MEF.
Experiments measuring HSV replication in isolated
individual cells (Table 4) confirm this to be the case;
however, the strain-related differences seen with single
cells were not as great as differences seen in MEF
monolayers. Virus yield on a per cell basis was also
higher in monolayers (Table 1) than in single cells (Table 4). These observations are consistent with those of
other investigators.1213
The data indicate that differences in virus production
by MEF monolayers from inbred mice are due to differences in post-adsorption events, rather than differences in the ability of the cells to adsorb virus or to
differences in the proportion of infectible cells present.
Therefore, host-determined restriction of virus production must involve virus penetration, uncoating,
transcription, translation, and/or assembly.
Differences in the level of interferon produced by
inbred mouse strains infected by Newcastle disease virus and HSV are known to exist. 1415 Although cells
cultured in vitro are isolated from cellular and humoral
mediators of the immune response, the cultured cells
might produce interferon after infection of HSV. Interferon, which is produced by virus-infected cells, acts
by the induction of antiviral protein in uninfected cells.
Thus, host-determined differences in virus production
by single isolated cells (Table 4) are unlikely to be due
to the action of interferon.
In humans, HSV may produce a relatively benign,
self-limited corneal epithelial infection or a more severe
stromal keratitis. It has been hypothesized that differences in the host immune response3"6 or genetically
determined virus factors216 account for these individual
differences in the severity of ocular disease produced
by HSV. Our results suggest that genetically determined
differences in the ability of host cells to support HSV
replication may also be important in determining the
subsequent course of infection.
Key words: herpes simplex, virus, inbred mice, replication,
fibroblasts, keratitis, BALB/c, C57BL/6
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914
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1986
Acknowledgment
The authors thank Ms. Janice C. Kindle for expert technical
assistance.
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