J. gen. Virol. (1989), 711,2487-2494. Printed in Great Britain
2487
Key words: HA V/hybrid cells/propagation
Propagation of Hepatitis A Virus in Hybrid Cell Lines Derived from
Marmoset Liver and Vero Cells
By M A S A H I K O A S H I D A , 1. H I D E N O R I H A R A , 2 H I D E O K O J I M A , 2
T O M O T E R U K A M I M U R A , 2 F U M I H I R O I C H I D A 2 AND
CHUYA HAMADA 1
1Department of Virology and 2The Third Department of Internal Medicine, Niigata University
School o f Medicine, AsahimachL Niigata 951, Japan
(Accepted 4 May 1989)
SUMMARY
To establish monkey liver cell lines with a high susceptibility to hepatitis A virus
(HAV), marmoset (Saguinus labiatus) liver cells were fused with Vero cells deficient in
hypoxanthine-guanine phosphoribosyltransferase and the resulting hybrid cells were
selected in HAT medium. Of four hybrid cell lines obtained (S. la/Ve-1 to -4), three
(S. la/Ve-1, -3 and -4) were equally susceptible to HAV infection. When inoculated
with a virus isolated from marmoset liver tissue (10~ liver tissue extract) or a faecal
virus (10~ stool extract) from a human hepatitis A patient, all susceptible cell lines
showed a significant elevation of viral antigen activity as seen in radioimmunoassay
and/or immunofluorescent antibody assays, at 4 to 6 weeks post-infection (p.i.) with the
liver-derived inoculum and at 6 to 8 weeks p.i. with the stool-derived inoculum. In
S. 1a/Ve-1 cells, a representative of the susceptible hybrid cell lines, full adaptation of
HAV (liver tissue virus concentrate) to cell culture was attained after four serial
passages. Thereafter, the virus grew to a plateau titre of 108.5 TCIDs0/ml at 7 days p.i.
in a growth experiment. The infected cells showed no cytopathic effects but eventually
a persistent infection was established when a saturated level of virus growth was
reached.
The first successful in vitro propagation of hepatitis A virus (HAV) was reported by Provost &
Hilleman (1979), using primary marmoset liver explant cultures and low passage number foetal
rhesus monkey kidney cells as the host cell systems. Several reports followed claiming efficient
propagation of HAV in a variety of cell cultures (for review, see Provost, 1984; Binn et al., 1984;
Bradley et al., 1984; Purcell et al., 1984; Vallbracht et al., 1984; Venuti et al., 1985; Anderson,
1987; Cromeans et al., 1987). However, most of these cell culture systems were inadequate.
Primary monkey kidney cell cultures were often contaminated with cytopathic viral agents
(Provost & Hilleman, 1979; Daemer et al., 1981; Binn et al., 1984). In addition, established
monkey kidney cell lines free from the contaminating agents were less susceptible to HAV
compared to their primary counterparts (Flehmig, 1980, 1981 ; Kojima et al., 1981 ; Locarnini et
al., 1981 ; Lemon et al., 1983; Binn et al., 1984; Bradley et al., 1984; Purcell et al., 1984; Venuti et
al., 1985). HAV was also propagated in human diploid cell lines, but the growth of HAV in these
cells was handicapped by a relatively longer latent period and lower virus yield (Flehmig et al.,
1981 ; Gauss-Miiller et al., 1981 ; Provost et al., 1982; Provost, 1984). Therefore, to facilitate the
in vitro propagation of HAV, it is necessary to develop additional cell culture systems without the
above drawbacks and which show a high susceptibility to HAV.
Preferential growth of HAV in liver tissue has commonly been observed in infected
chimpanzees and marmosets (for review, see Deinhardt & Deinhardt, 1984; Shibayama et al.,
1985; Karayiannis et al., 1986). Primate liver cells appear to be intrinsically susceptible to HAV
0000-8907 © 1989 SGM
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infection. In this study, we attempted to establish monkey liver cell lines with a high
susceptibility to HAV. The method employed was to fuse marmoset (Saguinus labiatus) liver cells
with Vero cells, an established cell line from African green monkey kidney cells (Yasumura &
Kawakita, 1963), to allow continual propagation of the liver cells in vitro. This experimental
strategy stemmed from the assumption that the hybrid cell lines obtained would be highly
susceptible to HAV because of their hepatocyte lineage, and that they would not harbour any
contaminating agents since the cell culture conditions used to select hybrid cells were quite
stringent against metabolically deficient cells.
Primary cultures of marmoset liver cells were prepared by a modification of the method used
by Nakamura et al. (1981 ). For cell dispersion, marmoset liver tissue removed under anaesthesia
was perfused with 0.05 ~ collagenase (Sigma type 1) in Hank's balanced salt solution pH 7.5
supplemented with 4 mM-CaC12 and 10 mM-HEPES at 37 °C for 15 min. Dispersed cells were
washed with DMEM (Dulbecco's modified Eagle's minimum essential medium) by three
centrifugations at 50 g for 1 min and cultivated in 25 cm 2 plastic tissue culture flasks (2 x 106
cells/5 ml/flask; Nunc) using DMEM containing 10~ foetal bovine serum (FBS; Gibco) at
37 °C in a CO2 incubator. After 2 days in culture, liver cell monolayers were overlaid with the
same number of Vero cells deficient in hypoxanthine-guanine phosphoribosyltransferase
( H G P R T - Vero cells), which had previously been selected from the original cell line (Yasumura
& Kawakita, 1963) in the presence of 8-azaguanine (Littlefield, 1964). The mixed cell cultures
were kept at 37 °C in a CO2 incubator for 12 h to promote cell adhesion. Cell fusion was induced
by successive soaking of the mixed cultures in 50, 25 and 12.5 ~ polyethylene glycol (Mr 1300 to
1600; Sigma) in DMEM for 1 min each, then washing with DMEM three times. Finally, the
mixed cell cultures were supplemented with HAT medium (DMEM containing 100 Ix~,lhypoxanthine, 0-4 ~tM-aminopterin, 16 ~tM-thymidine and 10~ FBS) and incubated in the same
environment for the growth of hybrid cells.
The resulting hybrid cell lines as well as H G P R T - Vero cells as a parental cell control were
examined for their susceptibility to HAV infection by three methods: (i) a screening test for their
capacity to isolate marmoset- and patient-derived viruses, (ii) serial passage of the virus and (iii)
a virus growth experiment. In each of these investigations, flask and coverslip cultures were
infected with a given virus inoculum in each experiment and growth of the virus was followed by
three concomitant assays: RIA (radioimmunoassay), IFA (immunofluorescent antibody assay),
and infectivity titration except for the screening test, where the infectivity titration was not
performed.
After cell fusion, four hybrid cell lines, S. la/Ve-1, -2, -3 and -4, each having grown initially
as single colonies in separate flasks, were obtained. All of these hybrid cell lines could easily be
subcultured by a conventional tissue culture method with a doubling time of about 3 days. The
morphology of these hybrids was intermediate between those of the parental cells manifesting a
polygonal contour and transparent nuclei. In addition, their modal chromosomal numbers at the
fifth and 50th subcultures (75/72, 82/80, 80/74 and 72/67 for S. la/Ve-1, -2, -3 and -4,
respectively) were distinct from those of the parental cells (46 and 93 for the marmoset liver and
H G P R T - Vero cells, respectively). All of the experiments described below were conducted
using the fifth to 50th subcultures of these cells.
First, the hybrid cell lines were screened for their susceptibility to marmoset- and patientderived HAVs by a virus isolation experiment (Table 1). The marmoset-derived HAV was
prepared as a 10 ~ liver tissue extract from a marmoset after intravenous infection with animalpassaged HAV. The patient-derived HAV was the original faecal virus isolated from a hepatitis
A patient and prepared as a 10~ stool extract as described elsewhere (Kojima et al., 1981).
When inoculated with the liver tissue virus (Table 1), HAV antigen was initially found by RIA
and IFA in S. la/Ve-1 cells (1174 in RIA, 1 + in IFA) at 2 weeks post-infection (p.i.), then in
S. la/Ve-3 (1417/1 + ) and -4 (17639/4+) cells at 4 weeks p.i. ; thereafter, the antigen activities
increased to maximal levels at 6 (36066/4+ for S. la/Ve-4) to 8 (41605/4+ for S. la/Ve-1;
20890/3 + for S. la/Ve-3) weeks p.i. With the same liver tissue virus, H G P R T - Vero cells also
expressed the HAV antigen at 8 weeks p.i., yet the titre was low (1262/1 +). When the faecal
virus was used as the inoculum (Table 1), the three hybrid cell lines again expressed the viral
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2489
Table 1. Susceptibility of hybrid cell lines and HGPRT- Vero cells to liver tissue H A V and
faecal HA V: expression of HA V antigen in hybrid cell lines
Cell line
f
Weeks of harvest
Liver HAV*
1
2
3
4
6
8
Faecal HAV*
Primary
isolation
Passage
A
S. I a/Ve-1
2
4
8
2
4
S. 1a/Ve-2
S. la/Ve-3
S. la/Ve-4
HGPRT- Vero
837t
1174
1106
5961
26752
41605
(-)~
(1+)
(1+)
(2+)
(3+)
(4+)
313
605
117
370
393
NT
(-)
(-)
~rr§
(-)
(-)
NT
992
1005
252
1417
6869
20890
(-)
(-)
(-)
(1+)
(2+)
(3+)
671
785
595
17639
36066
rcr
(-)
(-)
(-)
(3+)
(4+)
~rr
392
456
421
299
892
1262
661 t
wr
1993
8372
15674
(-)~
(2+)
(2+)
(3+)
(4+)
490
276
159
512
421
(-)
~r
(-)
(-)
(-)
370
334
1874
10543
17067
(-)
(-)
NT
(3+)
(4+)
464
NT
19474
3589
12877
(-)
(-)
(3+)
(2+)
(3+)
505
380
454
282
401
(-)
(-)
(-)
(-)
(-)
(1+)
(-)
(-)
(-)
(-)
(-)
* The liver tissue-derived virus used here is a descendant strain of the faecal HAV recovered from a patient
(T.T.) (Kojima et al., 1981) after four serial passages in marmosets. The liver tissue of the final animal was
prepared as a 10~ homogenate in 0.15 u-phosphate-buffered saline pH 7.2, followed by centrifugation at 10000
r.p.m, for 1 h. The resulting supernatants were used as the liver tissue virus solution. The faecal virus used is the
original HAV of the hepatitis A patient and was similarly prepared as a 10~ stool extract.
t RIA was performed using a HAVAB-M kit (Abbott Laboratories). Anti-IgM-coated beads were serially
reacted with anti-HAV IgM, the experimental infected cell culture extracts, and 125I-labelled anti-HAV
antibodies. The results are presented as mean c.p.m, values of duplicate samples. The cut-off value given by
uninfected specimens was 1010 for hybrid cell lines, and 942 for HGPRT- Vero cells.
:~An indirect method was used for IFA. Acetone-fixed coverslips were reacted initially with simian antiserum
(1:10) to HAV for 1 h, then with fluorescein isothiocyanate-conjugated anti-human antibodies (1:10; Boehringer
Mannheim) for 1 h. The figures in parentheses, 4 +, 3 +, 2 +, 1+ and -, indicate 100, 75, 50, 25 and less than 0-5
fluorescing cells in the infected coverslip cultures.
§ NT, Not tested.
antigen at 8 weeks p.i., but the titres differed between the cell lines. To confirm positive isolation
of faecal virus, all of these antigen-positive cell culture specimens were passaged into fresh
cultures of each cell line, resulting in m a r k e d elevations in viral antigen activity at 2 to 4 weeks
p.i. (15674/4+ for S . l a / V e - 1 ; 17067/4+ for S . l a / V e - 3 ; and 12877/3+ for S . l a / V e - 4 ) .
H G P R T - Vero cells did not express appreciable amounts of H A V antigen after infection with
the faecal virus, and S. la/Ve-2 cells remained negative for the viral antigen throughout the
experiment.
Since the screening test revealed three hybrid cell lines, S. la/Ve-1, -3 and -4 to be equally
susceptible to the marmoset- and patient-derived HAVs, S. la/Ve-1 cells were arbitrarily
selected as a representative of the susceptible cell lines and used in the serial passage and growth
experiments. In the serial passage experiments (Table 2), a 10-fold concentrate of the liver tissue
virus was used as the starting inoculum to accelerate the growth of the virus in cells. W h e n
S. 1a/Ve-1 cells were inoculated with the liver tissue virus concentrate (Table 2), expression o f the
H A V antigen ( R I A and I F A ) commenced at 1 week p.i. in the first passage. Antigen expression
increased gradually to the maximal level at 4 weeks p.i., when nearly 100K of cells showed
specific fluorescence in I F A . In the subsequent passages using the infected cell culture extracts
underlined in Table 2 as inocula, the time to attain the m a x i m a l antigen activity (in the order of
104 c.p.m, in R I A and 4 + score in I F A ) shortened from 4 weeks in the first passage to 2 weeks in
the second and third passages, and eventually to 1 week in the fourth passage and afterwards.
Virus infectivity titres in the infected cell culture extracts at 1 week p.i. were 105.8 and 107.3
TCIDs0/ml in the first and second passages, respectively, then rose to a maximal titre of 108.5
TCIDs0/ml in the fourth passage. This maximal range of virus titres (107.5 to 108"5) was
constantly m a i n t a i n e d in the subsequent passages. These findings, together with those of the
R I A and I F A , indicated that full adaptation of the liver tissue virus to cell culture was achieved
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Table 2. Serial passages of HA V in S. la/Ve-1 cells and H G P R T - Vero cells
HAV antigen expression at weeks p.i.
Cell line and
passage level
S. la/Ve-1
1st
2rid
3rd
4th
5th
6th
7th
8th
9th
10th
15th
20th
•
1
2
1539 (1 + ) t
5591 (2+)
5250 (1 +)
15516 (4+)
16856 (4+)
25397 (4+)
27555 (3+)
23 502 (4 +)
42502 (4+)
2__77883 (4+)
2_88198 (4+)
18206 (4 +)
2807
11283
13996
23294
20610
(2+)
(4+)
(4+)
(4+)
(4+)
ND
ND
ND
ND
ND
ND
ND
3
4
8330 (2+)
28824 (4+)
19248 (4+)
ND
24776 (4+):~
24872 (4+)
17723 (4+)
ND
Virus
~ infectivity (loglo
TCIDso/ml)*
5-8
7-3
ND§
8"5
ND
ND
ND
NO
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
7"5
ND
8"0
ND
8'5
8'5
8"0
822 (-)
722 (-)
3291 (1+)
11436 (3+)
15965 (3+)
1062 (1-~)
2042 (1+)
8266 (3+)
14350 (3+)
17221 (4+)
4.5
4-5
5.3
6.5
6-7
H G P R T - Vero
1st
2nd
3rd
4th
5th
261
540
672
1256
6954
(-)
(-)
(-)
(1 +)
(2+)
490
331
990
7661
7852
(-)
(-)
(-)
(2+)
(2+)
* Virus infectivity titres were assayed at 1 week p.i. by an endpoint dilution method combined with a solidphase enzyme immunoassay. Serial 10-fold dilutions (0.1 ml each) of cell culture extracts were inoculated into
S. 1a/Ve-1 cell cultures in 96-well microculture plates (five wells per dilution; Coming). After 2 h virus adsorption,
plates were supplemented with 0-1 ml/well DMEM containing 1~ FBS and incubated at 37 °C in a CO2 incubator
for 2 weeks. At the end of incubation, plates were washed, fixed with an 80~ methanol solution containing 0-03
hydrogen peroxide at 4°C overnight, and reacted with peroxidase-conjugated anti-HAV immunoglobulin
(Abbott) for 1 h, then with o-phenylenediamine solution for 30 min. Absorbance at 492 nm was determined for the
reaction mixtures; specimens giving over twice the mean value given by uninfected controls were taken as positive
for HAV antigen expression. TCIDs0 values were calculated according to the method of Reed & Muench (1938)
from the incidence of HAV antigen expression in the infected wells.
t For details of RIA, see Table 1.
Cell culture extracts giving the viral antigen activities underlined in the table were used as inocula for
subsequent passages.
§ ND, not done.
in the fourth passage. As c o m p a r e d with the results of S. 1a/Ve-I cells, serial passage of the liver
tissue virus concentrate in H G P R T - Vero cells resulted in less efficient growth of virus and
lower viral antigen expression (Table 2). F o r optimal expression of the viral antigen, it was
necessary to cultivate the infected cells for at least 4 weeks until the fifth passage and afterwards
(data not shown), and the virus infectivity titres at 1 week p.i. remained 10- to 100-fold lower
than those in S. la/Ve-1 cells at the corresponding passage levels.
In the viral growth experiment (Fig. 1), cells were inoculated with infected cell culture extract
in the 10th passage in Table 2 at an input m.o.i, of 100 TCIDso/cell. I n this experiment also,
S. la/Ve-1 cells far surpassed H G P R T - Vero cells in the efficiency of virus growth. In the
infected S. la/Ve-1 cells (Fig. 1 a), virus infectivity started to increase at 2 days p.i., then rose
steeply to a plateau titre of 10 s's T C I D s o / m l at 7 days p.i. Thereafter, this titre was m a i n t a i n e d
until 14 days p.i. Expression of H A V antigen as demonstrated by R I A and I F A followed a
similar pattern, giving plateau activities (2.5 × 104 to 3-0 x 104 c.p.m, and nearly 1 0 0 ~
fluorescing cells) at 7 days p.i. and thereafter. In H G P R T - Vero cells, on the other hand, the
infectious virus titre began to increase at 4 days p.i., gradually reaching a plateau at 10 days p.i.
when the titre remained 100-fold lower than that in S. 1a/Ve-1 cells. Likewise, the viral antigen
activity in this parental cell line was much lower, giving 1 0 ~ fluorescing cells in I F A and
approx. 103 c.p.m, in R I A as the optimum. During virus growth experiments, the infected
S. 1a/Ve-1 cells did not show any sign of c.p.e, and, when subjected to serial subcultures, the cells
grew as well as their uninfected counterparts. In addition, the plateau level of virus titres was
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2491
9
. . . . . .
'
~ - - & . _ ' _ ~
i
100
30
-25 ~
80
0
60
6
"~
40
20
~4
~9
i
i
|
|
i
i
i
i
l
i
i
I
i
0
0
30
100 :~
"~
(b)
25 "~
"~
8
)<
80
<
60
>
d~
6
.~
. 40
5
~
20
4
0
1
2
3
4
5
6
7
8
9
Time p.i. (days)
10
12
14
0
Fig. 1. Growth of HAV in S. la/Ve-1 cells (a) and HGPRT- Vero cells (b). The virus inoculum used was
the infected cell culture extracts from the 10th passage of HAV in Table 2. Flask and coverslip cultures
of S.la/Ve-1 ceils and HGPRT- Vero cells were inoculated with the extracts (0.5 ml and 0.2 ml
respectively)at an m.o.i, of 100 TCIDso/cell. Infected cultures were kept at 37 °C in a CO2 incubator for
2 h for virus adsorption, washed three times with DMEM, supplemented with the same medium
containing 170FBS, and further incubated in the same environmentfor virus growth. At daily intervals
thereafter, expression of the virus and viral antigen in the infected cultures were monitored by
infectivity titration (It), RIA (0) and IFA (O) as described in Tables 1 and 2.
continually maintained, indicating that a persistent infection of cells at a saturated level of virus
growth resulted (data not shown).
I n the present study, marmoset liver cells were fused with Vero cells to provide cell lines with a
high susceptibility to H A V infection. The reason for the use of Veto ceils as the partner cell line
was their known, though restricted, susceptibility to H A V infection (Kojima et al., 1981;
Locarnini et al., 1981) and their species homology with marmoset liver cells, from which genetic
stability of the hybrid cell lines was anticipated. In fact, marmoset liver cells fused with a cell
line of a distinct species (Chinese hamster ovary cells) were also susceptible to HAV, but only
transiently in the early subcultures (unpublished observation).
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Table 3. Comparison o f virus yields from several HA V-infected primate cell cultures
Cell and
cell line*
AGMK
FRhK6
HDEF
HFS
BS-C-1
HGPRT- Vero
S. la/Ve-1
Maximal virus yield
(TCIDso/ml)
107 to
10a
107 to
106 to
105 to
106.5
108`s
109
108
107
107
Reference
Purcell et al. (1984)
Provost (1984)
Provost (1984)
Vallbracht et al. (1984)
Binn et aL (1984)
This paper
This paper
* AGMK, African green monkey kidney cells; BS-C-1, African green monkey kidney cell line; FRhK6, foetal
rhesus monkey kidney cells; HDEF, human diploid embryo fibroblasts; HFS, human embryo fibroblast strain.
Growth of the hybrid cell lines established here was not inhibited by H A T medium. Their
modal chromosome numbers were clearly distinct from those of the parental cells and cell line.
Morphologically, the hybrid cell lines resemble the parental liver ceils since they show polygonal
contours and transparent nuclei. In all of the four hybrid cell lines, albumin production was also
evident by IFA in their early subcultures approximately up to the 10th passage (data not shown).
Although it is circumstantial, these observations indicate that these cell lines are genuine
hybrids. Chromosome analysis including G-banding, which was repeatedly applied to the
hybrid cell lines to prove their authenticity directly, was not determinative, because of the lack
of convincing markers to distinguish the parental cell chromosomes.
Three out of the four hybrid cell lines obtained were equally susceptible to the marmoset liver
and human faecal viruses. In S. 1a/Ve-1 cells, a representative of these cell lines, full adaptation
of the liver tissue virus to cell culture was attained in the fourth passage. Subsequently, the cell
culture-adapted virus grew constantly to maximum titres of 107.5 to 108.5 TCIDs0/ml at 7 days
p.i. For the maximum yield of cell culture-adapted HAVs, S. 1a/Ve-1 cells were compared with
other cells and cell lines that have been reported to be susceptible to HAV infection. Table 3
shows these results. The highest virus yield was obtained in African green monkey kidney cells
(107 to 109) and the lowest in BS-C-1 cells (105 to 107). In a review article (Provost, 1984),
FRhK6 cells were considered to be one of the most HAV-susceptible cell types. A G M K ,
FRhK6, and S. la/Ve-1 cells are comparable in maximal virus yield (107 to 109, 108 and 108.5
each) in this Table. According to these comparisons, the susceptible hybrid cell lines, S. 1a/Ve-1
at least, appear to be ranked amongst the most sensitive to HAV. This sensitivity of S. la/Ve-1
cells to HAV may depend on the hepatocyte lineage of the cell line, since H G P R T - Vero cells,
the parental partner for cell fusion, and the original Vero cells (Kojima et al., 1981 ; Locarnini et
al., 1981) were much less sensitive to infection by the same strain of HAV.
In the S. la/Ve-1 cell line, virus isolation using early subcultures (fifth to 10th passages)
resulted in an efficient recovery of the virus derived from liver tissue and from faeces (Table 1).
Growth of HAV in later subcultures (10th to 30th passages) in the same cell line was substantial,
yielding a high infectivity titre of 108.5 TCIDs0/ml (Fig. 1). Advanced subcultures (40th to 50th
passages) of S. la/Ve-1 cells were also used for an epidemiological study of virus isolation from
faecal specimens of patients (Table 4; details to be published elsewhere). Recovery of faecal
HAVs by this cell line was far more efficient than by other primate cell lines, though some faecal
specimens were omitted from the survey of the latter cell lines. It should be noted that the hybrid
cell line could isolate HAVs even from specimens seemingly negative for viral antigen in RIA.
Thus S. la/Ve-1 cells are susceptible to diverse strains of HAV and are able to withstand
multiple subculture passages.
It was anticipated that each hybrid cell line would have 139 chromosomes (46 from the
marmoset cells plus 93 from the Vero cells). However, the modal chromosome numbers of cells
examined at the fifth and 50th subcultures were 75/72, 82/80, 80/74 and 72/67 for S. la/Ve-1, -2,
-3 and -4 cells, respectively. This means that the hybrid cell lines had undergone major
chromosomal deletions before the fifth passage, but are genetically stable.
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2493
Table 4. Primary isolation of HA V from faecal specimens of patients using various monkey cell
lines
Detection of H A V antigen in cell linest
A
Inoculum (c.p.m.)*
S. la/Ve-1
BS-C-1
LLC-MK2
BGM
H G P R T - Vero
T.T. (11356)
1450 (4)
12561 (8)
NT
NT
NT
--:~
_
-
NT
rcr
N'r
~rr
NT
NT
-
-
-
-
_
_
-
-
-
-
Y.T. (11446)
K-5 (12816)
Ku-1 (247)
Ku-2 (135)
Ku-3 (8518)
Ku-4 (11652)
7327
7327
6911
28072
(6)
(6)
(4)
(6)
869
13880
1094
14519
(2)
(8)
(2)
(8)
911
6770
1045
9423
(2)
(8)
(2)
(6)
4360 (4)
4360 (4)
_
-
* The T.T. and Y.T. inocula were obtained during an outbreak of hepatitis A in Kyoto during 1978. The K-5
inoculum came from a sporadic case of hepatitis A which occurred in Niigata in 1983. The Ku-1 to -4 inocula were
collected in Chikugo region from 1979 to 1986. All inocula were prepared as 5 to 10 % faecal homogenates clarified
by centrifugation at 10000 r.p.m, for 30 rain.
i" H A V antigen activities are presented as c.p.m. The cut-off value was 823. Results are shown as initial c.p.m.
significant (incubation period, weeks) and m a x i m a l c . p . m obtained (incubation period, weeks).
~: C.p.m. not significant during 8 weeks incubation.
In conclusion, the experimental strategy followed in this study was effective, yielding three
hybrid cell lines from marmoset liver cells with a high susceptibility to HAV infection. These
hybrid cell lines, in addition, remained intact through subculture passages without manifesting
any cytopathic changes as were often encounted in the primary cultures of monkey kidney cells.
The experimental strategy described here is worthy of consideration for investigations of other
hepatitis viruses that are currently difficult to propagate in vitro.
We would like to t h a n k Drs M. Sata and K. Tanigawa (The Second D e p a r t m e n t of Internal Medicine, K u r u m e
University, Japan) for kindly providing patients' faecal specimens. Part of this work was supported by a grant
from the Ministry of Health and Welfare, Japan.
REFERENCES
ANDERSON, D. A. (1987). Cytopathology, plaque assay, and heat inactivation of hepatitis A virus strain HM175.
Journal of Medical Virology 22, 35-44.
BINN, L. N., LEMON, S. M., MARCHWICKI,R. H., REDFIELD, R. R., GATES, N. L. & BANCROFT,W. H. (1984). Primary
isolation and serial passage of hepatitis A virus strains in primate cell cultures. Journal of Clinical Microbiology
20, 28-33.
BRADLEY, D. W., SCHABLE, C. A., McCAUSTLAND,K. A., COOK, E. H., MURPHY, B. L., FIELDS, H. A., EBERT, J. W.,
WHEELER, C. & MAYNARD, I. E. (1984). Hepatitis A virus. Growth characteristics of in vivo and in vitro
propagated wild and attenuated virus strains. Journal of Medical Virology 14, 373-386.
CROMEANS, T., SOnSEY, M. D. & FIELDS, n. A. (1987). Development of a plaque assay for a cytopathic, rapidly
replicating isolate of hepatitis A virus. Journal of Medical Virology 22, 45-56.
DAEMER, R. J., FEINSTONE, S. M., GUST, I. D. & PURCELL, R. H. (1981). Propagation of h u m a n hepatitis A virus in
African green monkey kidney cell culture: primary isolation and serial passage. Infection and Immunity 32,
388-393.
DEINHARDT, F. & D E I ~ D T , J. B. (1984). Animal models. In Hepatitis A, pp. 185-204. Edited by R. J. Gerety.
Orlando & London. Academic Press.
FLEHmG, B. (1980). Hepatitis A virus in cell culture. I. Propagation of different hepatitis A virus isolates in a fetal
rhesus monkey kidney cell line (FrhK-4). Medical Microbiology and Immunology 168, 239-248.
FLEHMIG, n. (1981). Hepatitis A virus in cell culture. II. Growth characteristics of hepatitis A virus in F r h K - 4 / R
cells. Medical Microbiology and Immunology 170, 73-81.
FLI/HMIG, B., VALLBRACHT,A. & WURSTER,G. (1981). Hepatitis A virus in cell culture. I II. Propagation of hepatitis A
virus in h u m a n embryo kidney cells a n d h u m a n embryo fibroblast strains. Medical Microbiology and
Immunology 170, 83-89.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:23:13
2494
Short communication
GAUSS-MULLER,V., FROESNER,G. G. & DEINHARDT,F. (1981). Propagation of hepatitis A virus in human embryo
fibroblasts. Journal of Medical Virology 7, 233-239.
KARAYIANNIS, P., ENTICOTT, J. M., MOORE, D., PIGNATELLI, M., BRENES, F., SCHEUER, P. J. & THOMAS, H. C. (1986).
Hepatitis A virus replication in tamarins and host immune response in relation to pathogenesis of liver cell
damage. Journal of Medical Virology 18, 261-276.
KOJIMA, H., SHIBAYAMA, T., SATO, A., SUZUKI, S., ICHIDA, F. & HAMADA, C. (1981). Propagation of human hepatitis A
virus in conventional cell lines. Journal of Medical Virology 7, 273-286.
LEMON,S. M., m~rN,L. N. &Wa~CHWICKI,R. n. (1983). Radioimmunofocus assay for quantitation of hepatitis A virus
in cell cultures. Journal of Clinical Microbiology 17, 834-839.
Lxr'rLEFIELD, S. W. (1964). Three degrees of guanyl acid inosinic acid pyrophosphorylase deficiency in mouse
fibroblasts. Nature, London 203, 1142-1144.
LOCARNINI, S. A., COULEPIS, A. G., WESTAWAY, E. G. & GUST, I. D. (1981 ). Restricted replication of human hepatitis A
virus in cell culture. Intracellular biochemical studies. Journal of Virology 37, 216-225.
N~,MURA, Y., AOYAMA,K. & ICHIH~*,A, A. (1981). Methods of preparation and primary culture of isolated
hepatocytes. Protein, Nucleic Acid and Enzyme 24, 55-76 (in Japanese).
PROVOST,v. J. (1984). In vitro propagation of hepatitis A virus. In Hepatitis A, pp. 245-26 i. Edited by R. J. Gerety.
London. Academic Press.
PROVOST,P. J. & HILLEMAN,M. R. (1979). Propagation of human hepatitis A virus in cell culture in vitro. Proceedings
of the Society for Experimental Biology and Medicine 160, 213-221.
PROVOST, P. J., McALEER, W. J. & HILLEMANN, M. R. (1982). In vitro cultivation of hepatitis A virus. In Viral Hepatitis,
pp. 21-30. Edited by W. Szmuness, H. J. Alter & J. E. Maynard. Philadelphia: Franklin Institute Press.
PURCELL,R. H., FEINSTONE,S. M., TICErIURST,J. R., DAEMER,R. J. & B~OUDY, B. M. (1984). Hepatitis A virus. In Viral
Hepatitis and Liver Disease, pp. 9-22. Edited by G. N. Vyas, J. L. Dienstag & J. H. Hoofnagle. Orlando:
Grune & Stratton.
REED, L. J. & MUENCrl, H. (1938). A simple method of estimating fifty per cent endpoints. American Journal of
Hygiene 27, 493497.
SHIBAYAMA, T., KOJIMA, H., ASHIDA, M., HIROSE, S., SATO, A., KAMIMURA, T., HAMADA, C., SHIMIZU, S. & ICHIDA, F.
(1985). Localization of hepatitis A virus in marmoset liver tissue during the acute phase of experimental
infection. Gastroenterologia Japonica 20, 564-572.
VALLB~crrr, A., HOF~a~,~, L., WURSTER,K. G. & FLm~MIG,B. (1984). Persistent infection of human fibroblasts by
hepatitis A virus. Journal of General Virology 65, 609-615.
VENUTI, A., DI RUSSO, C., DEL GROSSO, N., PATI'I, A.-M., RUGGERI, F., DE STASIO, P. R., MARTINIELLO, M. G., PAGNOTTI,
P., DEGENER, A. M., MIDDULLA, M., PANA, A. & PEREZ-BERCOFF, R. (1985). Isolation and molecular cloning of a
fast-growing strain of human hepatitis A virus from its double-stranded replicative form. Journal of Virology
56, 579-588.
Y~UMURA,Y. & I¢~W~d~ITA,A. (1963). Study of SV40 by tissue cultures. Nippon rinsho 21, 1201-1219 (in Japanese).
(Received 6 February 1989)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:23:13