403
J. gen. Virol. (1981), 54, 403-407
Printed in Great Britain
Varicella-Zoster Virus Infection of Diploid and Chemically Transformed
Guinea-pig Embryo Cells: Factors Influencing Virus Replication
(Accepted 29 January 1981)
SUMMARY
Factors influencing the replication of varicella-zoster virus (VZV) in guinea-pig
embryo cells were evaluated using both diploid cells (GPEC) and a chemically
transformed cell line (GPT). Wild-type and vaccine strains of VZV were successfully
isolated and serially propagated in GPEC prepared from early gestation whole
embryos (<2 cm in length). Low passage GPEC (_<5 subcultivations) were more
susceptible to VZV infection than high passage GPEC (>5 subcultivations), and
guinea-pig cells were consistently less permissive than human diploid cells. Cell-free
virus was produced from VZV-infected GPEC cultures by sonication and peak
yields of 103 p.f.u./ml were obtained. In addition, we report the isolation and
propagation of VZV, as well as production of cell-free virus, in GPT. Both GPEC
and GPT ceils were less susceptible to VZV infection than human cells. However,
viral replication was enhanced by incubation of VZV-infected GPT cultures at 32 ° C
rather than 36 °C.
Guinea-pig embryo cells (GPEC) are the only non-primate cell type in which the growth of
varicella-zoster virus (VZV) has been established (Fioretti et aL, 1973; Harbour & Caunt,
1975). However, both the comparative susceptibility of GPEC versus human cells to infection
with VZV (S61zt-Sz6ts, 1964, 1965; Sefcovicova, 1971) and the capacity of VZV-infected
GPEC to yield infectious cell-free virus (Fioretti et al., 1973; Harbour & Caunt, 1975)
remain controversial. To further characterize the growth of VZV in GPEC, we have
evaluated the susceptibility of GPEC to infection with wild-type and vaccine strains of VZV,
compared the growth kinetics of VZV in both guinea-pig and human cells, and measured
yields of cell-free virus prepared from VZV-infected GPEC. In addition, we report the
isolation and propagation of VZV in a chemically transformed line of guinea-pig cells (GPT).
Primary and secondary GPEC cultures were prepared by standard methods (Kuchler,
1977) from early gestation embryos measuring 1.0 to 1.5 cm in length. Cells were grown at
36 °C in Eagle's minimal essential medium (MEM) supplemented with penicillin (100 u/ml),
streptomycin (100/~g/ml) and 12 % heat-inactivated (56 °C for 30 min) foetal bovine serum
(FBS). Confluent cell monolayers were harvested using trypsin-EDTA and subcultured at a
1:3 split ratio. The GPT was established by Evans & DiPaolo (1975) by cloning a single cell
derived from strain 2 guinea-pig embryo cultures previously treated with benzo(a)pyrene.
GPT cells (obtained at passage 23 from the American Type Culture Collection, Rockville,
Md., U.S.A.) were grown at 36 °C in supplemented MEM with 10% FBS and were
subcultured at a i : 3 split ratio. The following cell-free virus strains were used in this investigation: vaccine strain VZV-Oka, isolated in human embryonic lung fibroblasts (HELF) and
subsequently passaged in HELF 11 times, GPEC 12 times, and human diploid cells (WI-38)
6 times (Takahashi et al., 1975); wild-type strains of VZV obtained from vesicle fluids
collected from patients with early chickenpox or herpes zoster infection; and strain VZV-32,
isolated and serially propagated in human melanoma cell cultures at an incubation
temperature of 32 °C (Grose & Brunell, 1978). All virus strains were serially passaged by
harvesting infected monolayers with trypsin-EDTA, resuspending cells in supplemented
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MEM with 10% FBS and inoculating infected cell suspensions on to nearly confluent
monolayers at a ratio of 1 infected cell monolayer to 3 uninfected cell monolayers. Infected
GPEC cultures were incubated at 36 °C. However, to slow down cell growth in virus-infected
transformed cells, infected GPT cultures were incubated at 32 ° C.
The susceptibility of GPEC for the isolation of wild-type strains of VZV was evaluated
using vesicle fluid samples obtained from five patients within 48 h of the onset of chickenpox.
VZV was isolated in low-passage GPEC cultures (< 5 subcultivations), as well as in control
HELF cultures, from all five vesicle fluid samples. The cytopathic effect (c.p.e.) observed in
both GPEC and H E L F monolayers was characterized by foci of rounded refractile ceils.
After two to six serial passages in GPEC cultures, viral isolates were identified as VZV by
co-cultivation of infected GPEC with HELF and subsequent examination by the fluorescent
antibody to membrane antigen (FAMA) assay (Williams et al., 1974). Using human VZV
immune serum (FAMA titre 1:1024), VZV-specific fluorescence was observed on infected
cell membranes, whereas human non-immune serum (FAMA titre < 1:2) failed to produce
immunofluorescence. In addition, large intranuclear inclusions (Cowdry type A) were
observed in infected GPEC stained with haemotoxylin and eosin. Neither cytopathic changes
nor inclusions were seen in uninfected GPEC cultures.
The propagation of VZV in GPEC cultures was evaluated using one wild-type strain
isolate and the vaccine strain (VZV-Oka). No changes in the character of c.p.e, were
observed through 15 and 20 serial passages in GPEC cultures of a wild-type strain and the
vaccine strain respectively. Spontaneously released virus could not be detected in the media
from cultures showing 75 % c.p.e, by inoculation onto HELF or GPEC monolayers, but
cell-free virus was obtained by sonic disruption of infected GPEC. Serial passage did not
affect the virus titre in a systematic way. The kinetics of virus growth was determined by
harvesting GPEC monolayers at 24-h intervals after inoculation with trypsin-dispersed
VZV-Oka P9-infected GPEC cells. At harvest, the cultures were examined for c.p.e. The cells
were scraped into 10% sorbitol in 0.05 u-tris-HC1 pH 7.4, 0-001 M-MgC12, and disrupted
with a Branson Sonifier (model W185D with a microtip). Cell free virus titres were
determined on both low-passage GPEC and HELF cultures by the virus plaque assay
described by Grose & Brunell (1978). The c.p.e, observed at 24 h, 72 h and 96 h was 30%,
75% and 85% respectively. The respective virus titres determined on GPEC cells were 5
p.f.u./ml, 480 p.f.u./ml and 355 p.f.u./ml and as determined on HELF cells were 76 p.f.u./ml,
1760 p.f.u./ml and 700 p.f.u./ml. As expected, at each interval the titres determined on H E L F
cells were higher than those obtained on GPEC cultures, but the maximum titre in each case
was obtained at 72 h post-infection.
These results were extended by comparing the efficiency of plating of cell-free virus on
HELF cells to cultures of GPEC at different passage numbers. Nearly confluent cultures of
GPEC at 2, 5, 7 or 10 subcultivations, or HELF at 27 subcultivations, in 16 mm tissue
culture wells, were inoculated in quadruplicate with cell-free VZV-Oka P11 from GPEC
cultures. The virus titres were determined by virus plaque assay. Fig. 1 shows that, as
expected, the highest titres were obtained on HELF cells. The highest titre on GPEC cultures
were obtained on passage 5 cells, while no plaques were observed after 10 subcultivations.
Skin-muscle and lung cultures from guinea-pig embryos greater than 2 cm in length were also
found to be less susceptible to virus infection than less differentiated whole embryo (< 2 cm)
cell cultures (data not shown).
Similar tests were carried out using the chemically transformed GPT line. VZV-Oka and
VZV-32 strains were serially propagated in GPT cells at 32 °C and wild-type strains from
one early chickenpox patient and two early zoster patients were successfully isolated
and serially propagated 8 times in GPT cells. All infected cell cultures displayed a
predominantly syncytial c.p.e, and electron microscopy demonstrated herpesvirus-like naked
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Fig. 1. Efficiencyof plating cell-free VZV in human versus guinea-pigembryo cell (GPEC) cultures. A
plaque assay was performed with replicate aliquots of cell-free virus (VZV-Oka P l l in GPEC)
inoculated on to the followingcell monolayers: 1, human embryonic lung fibroblasts (HELF P27); 2,
low passage (P5) GPEC; 3, low passage (P2) GPEC; 4, high passage (P7) GPEC; 5, high passage
(P10) GPEC. Note: no plaques were observed in GPEC P10 in which the virus titre was < 2.5 p.f.u./ml.
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Fig. 2. Efficiencyof plating GPT<terived VZV at 32 °C versus 36 °C. Four 75 cm2 GPT monolayers
were infected with VZV-32 (P8 in GPT culture at 32 °C), incubated at 32 °C and harvested on days 3,
5, 7 and 9 post-infection. Sonically released cell-free virus was titrated on duplicate sets of HELF
monolayers incubated at 32 °C (0) and 36 °C (O). After 6 days incubation, monolayers were fixed
and stained, and plaques were counted. Virus titre is expressed as yield per 75 cm2 monolayer (p.f.u./
2 ml sonicate).
and enveloped particles near nuclear and plasma membranes. Cell-free virus was detected
only after sonic disruption. The efficiency of plating V Z V - O k a P4 in G P T cultures was
evaluated using replicate aliquots of cell-free virus simultaneously titrated at 32 ° C on H E L F
(passage 16), h u m a n m e l a n o m a and G P T monolayers, and peak titres of 6350, 45 and
30 p.f.u./ml respectively were obtained. The effect of temperature on cell-free virus yields
was also determined. Fig. 2 shows that GPT-propagated VZV plated twice as efficiently on
H E L F cells at 32 ° C than at 36 °C. In additional experiments, the relative replication of
VZV on G P T cultures at 32 ° C and 36 ° C was determined. Monolayer cultures were
inoculated with equal amounts of VZV-infected G P T cells and, after adsorption, incubated
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at either 32 °C or 36 °C. Cell-free virus was harvested and titrated on HELF cells at 32 °C
and 36 °C at 3, 5 and 7 days post-infection. Cell numbers were determined from parallel
uninfected cultures. At 36 °C, cell doubling occurred about every 36 h, whereas over a 4 day
period at 32 °C, cell numbers barely doubled. At 36 °C a peak titre of 3.67 × 103 p.f.u./ml
was observed at 5 days post-infection and the monolayers contained 1.96 × 10 7 cells. At
32 °C, the virus titre was 6.67 × 103 p.f.u./ml and the cell number was 7 × 106. The crude
efficiency of replication (as determined by p.f.u./cell) was therefore five times higher at the
lower temperature.
Our study confirms the original observation of S61tz-Sz6ts (1964, 1965) that virus present
in vesicle fluid can be isolated in cell cultures derived from early-gestation whole guinea-pig
embryos. Our finding that titres of GPEC-derived cell-free virus were consistently higher on
HELF cultures is also consistent with that of Harbour & Caunt (1975), who reported that
GPEC were threefold less sensitive to an infected HELF inoculum, and 1000-fold less
sensitive to cell-free VZV from HELF cultures. The inability of Sefcovicova (1971) to
duplicate S61tz-Sz6ts experiments may have been a consequence of Sefcovicova's preparation
of skin-muscle cultures from mid to late gestation guinea-pig embryos (their length was not
stated) rather than using less differentiated whole embryonic tissue which we found to be
more sensitive. Differentiation of the guinea pig foetus is essentially complete by day 35 of
gestation (term 68 days) when the foetus is approx. 2 cm long (Draper, 1920).
In contrast to Harbour & Caunt (1975), Fioretti et al. (1973) found GPEC and a human
fibroblast cell (WI-38) to have comparable sensitivities to VZV infection. More recently,
Myers et al. (1980) reported the adaption of VZV to GPEC culture after serial passage in
human foreskin fibroblasts (HFF), Hep-2 ceUs and low-passage GPEC cells. Virus titres were
higher in H F F than GPEC cultures before passage in Hep-2 cells, but after adaption comparable infectivities were observed in HFF, Hep-2 and GPEC cultures. Both Harbour & Caunt
(1975) and Myers et al. (1980) prepared GPEC from whole embryos < 2 cm whereas Fioretti
et al. (1973) used skin-muscle cultures from 4-week-old embryos and passage numbers
between 1 and 50. Taken together, the results suggest that both the gestational status of the
guinea-pig embryos and the cell passage number are critical factors influencing VZV
replication in GPEC cultures.
The susceptibility of chemically transformed guinea-pig embryo cells to infection with VZV
has some important implications. As a continuous cell line, GPT cells obviate the need for
repeated preparation of primary early gestation embryo cell cultures. In addition, Viral
antigens prepared from VZV-infected strain 2 guinea-pig-derived GPT cultures could be used
to immunize strain 2 guinea-pigs, with the resultant antisera being devoid of antibody against
either human or guinea-pig cellular determinants. Thus, the inbred strain 2 guinea-pig could
serve as an alternative to the vervet monkey for production of virus-specific antibody
(Harbour & Caunt, 1979).
This research was supported by awards from the Jeane B. Kempner Foundation and the Southern
Medical Association Project Fund to Betty J. Edmond. Charles Grose is a recipient of Young
Investigator Research Grant 14604 from the National Institute of Allergy and Infectious Diseases. We
gratefully acknowledge the critical review of Dr John D. Shanley and Dr Martin G. Myers during the
preparation of this manuscript.
Virology Laboratory
Department of Pediatrics
University of Texas Health Sciences Center
San Antonio, Texas 78284, U.S.A.
BETTY J. EDMOND*t
CHARLES GROSE
PHILIP A. BRUNELL
~"Present address: Departmentof Pediatrics,Universityof Iowa, Iowa City, Iowa 52242, U.S.A.
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407
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