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Key words: Newcastle disease virus/interferon/L cells~limitedgrowth
Relation of Interferon Production to the Limited Replication of
Newcastle Disease Virus in L Cells
B y Y O S H I Y U K I N A G A I , 1. Y A S U H I K O I T O , 2
MICHINARI
H A M A G U C H I , 1 T E T S U Y A Y O S H I D A 1 AND
TOSHISADA MATSUMOTO 1
1Cancer Research Institute and 2Germ-Free Life Institute, Nagoya University School of
Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan
(Accepted 13 February 1981)
SUMMARY
Growth of Newcastle disease virus (NDV) in L cells, where the virus undergoes
limited replication, has been compared to that in fully permissive BHK-21 host cells.
The synthesis of viral proteins and the production of infectious progeny were found
to occur normally at early times of infection in L cells. However, the subsequent
amplification of viral protein synthesis did not take place and instead of infectious
virions, non-infectious haemagglutinin was the predominant product. This shift of
replication pattern from complete to incomplete virus production seemed to be
temporally related to the appearance and accumulation of interferon (IFN) in the
system. The addition of specific antiserum against mouse IFN to the infected L cell
cultures was able to circumvent such restriction of virus growth. In the presence of
the antiserum, synthesis of viral proteins was found to progress normally, with the
production of a high amount of infectious progeny comparable to that of the
permissive system. These results suggest that the limited replication of NDV in L cells
may be due to interference by the endogenously produced IFN during the course of
infection.
INTRODUCTION
Infection of L cells with Newcastle disease virus (NDV) results in limited replication of the
virus with reduced infectivity (Wilcox, 1959). The exact mechanism of this limited replication
is still unknown. Although the high yield of interferon (IFN) and the low yield of infectious
progeny in this virus-cell system seemed to be causally related, earlier studies have suggested
that this was unlikely. Youngner & Scott (1968) found that the virus yield did not increase
when IFN production was suppressed by actinomycin D. Furthermore, Thacore & Youngner
(1969) reported that NDVpi, which had been recovered from persistently infected L cells,
could grow well in L cells although it induced much higher amounts of IFN than the wild-type
NDV o. They suggested that this was additional evidence for the lack of relationship of IFN to
the limited virus growth.
We have also examined the influence of suppression of IFN production by actinomycin D
on virus growth in our L cell-NDV system. However, the data obtained were difficult to
interpret because of a non-specific loss in cellular viability. Therefore, in order to establish the
role of IFN more firmly, it was necessary to employ some other procedures that might exert a
more selective effect on the production and/or action of IFN. In this respect, use of specific
antiserum against IFN has been demonstrated to be valuable (Inglot et al., 1973; Vilcek et aL,
1977; Sekellick & Marcus, 1978). We found that administration of anti-mouse IFN serum to
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Y.
NAGAI
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the L ceU-NDV system overcame the restriction of virus growth, yielding a high amount of
infectious progeny comparable to a permissive system analysed in parallel. Thus, we provide
evidence that limited NDV growth in L cells is due to interference caused by endogenously
produced IFN.
METHODS
Virus and cells. Strain Miyadera of NDV was grown in the allantoic cavity of 11-day-old
chick embryo and the infectious aUantoic fluid was used as stock virus. L cells were grown in
minimum essential medium (MEM) containing 10% calf serum and BHK-21 cells in the same
medium supplemented with 10% tryptose phosphate broth.
Infectivity and haemagglutination titrations. The assay of virus titre by plaque assay on
monolayers of chick embryo cells and the haemagglutination (HA) test have been described
previously (Nagai, 1973).
Assay of interferon. IFN titres were determined using vesicular stomatitis virus (VSV) as a
challenge virus by the procedure of Shimokata et al. (1976). The monolayers of L and
BHK-21 cells were treated for 24 h with the appropriate serially diluted culture media of the
respective species of cells, infected with NDV and then challenged by 100 TCID50 of VSV.
After 3 days the development of cytopathic change was scored and IFN titre was expressed
by the reciprocal of the highest dilution at which cytopathic change was inhibited. NDV
contained in the samples was neutralized by anti-NDV serum before IFN titration.
Infection and sampling. Monolayers of L and BHK-21 cells grown in plastic Petri dishes
(diam. 35 mm) or in multi-weU tissue culture plates (24 wells; Falcon, Oxnard, Ca., U.S.A.)
were infected with NDV at different multiplicities. After incubation at 37 °C for 60 min, to
allow virus penetration, the cells were washed 5 times with MEM and after incubation in the
same medium for appropriate periods, were harvested by scraping into the medium. Before
virus titration the cells were subjected to three cycles of freezing and thawing.
Labelling of cells and analysis of viral protein synthesis. The infected cells were maintained
with MEM lacking methionine. Either anti-mouse IFN rabbit serum or normal rabbit serum
was added to the culture medium 1 h after infection to give a finalconcentration of 10%. At
every hour after infection the cells were labelled for a period of 1 h with [35S]methionine
(5 #Ci/ml). The labelled proteins were analysed by electrophoresis on 10% polyacrylamide
slab gels (Laemmli, 1970) and detected by autoradiography on a Kodak X-omat film.
Antisera. Anti-IFN serum was obtained through the courtesy of Dr S. Kono, National
Institute of Health, Tokyo, Japan. The antiserum was prepared by immunizing rabbits with
purified IFN induced in L cells by NDV. The antigen had a specific activity of about 107
international units IFN/mg. The anti-IFN serum had a capacity to neutralize 1280 units IFN
at a dilution of 1/10 under our experimental conditions. The anti-L cell serum was prepared
by immunizing rabbits with 1 x 108 to 8 x 108 L cells (Y. Ito, H. Aoki, Y. Kimura, K.
Shimokata & K. Maeno, unpublished results). Antiserum against NDV was prepared as
described previously (Nagai, 1973).
Radioisotope. L-[35S]methionine (400 Ci/mmol) was purchased from New England
Nuclear.
RESULTS
Growth characteristics of ND V in L cells
The single cycle growth of NDV in L cells has been characterized by low yield of infectious
progeny and synthesis of non-infectious haemagglutinin (Wilcox, 1959). We analysed first the
growth of NDV in L cells and compared the growth pattern to that in a fully permissive host,
BHK-21 cells. The infection was initiated by inoculation of the stock virus at an input
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Limited N D V growth and interferon
8
6.
I
I
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111
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<20 1-~-, . . . . ~ .... r - 7 .... i - - ~ .... ? 7
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Time after infection (h)
Fig. 1
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Time after infection (h)
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Fig. 2
Fig. 1. Kinetics of virus growth and IFN production in BHK-21 (O) and L ( 0 ) cells infected with
NDV at an input multiplicity of 10 p.f.u./cell. Infectivity (p.f.u.) (
) and IFN titres ( - - . - - ) were
determined as described in Methods. HA was also determined with each sample and the ratios of
p.f.u. : H A units ( - - - ) are shown.
Fig. 2. Growth of N D V in BHK-21 (O) and L (O) cells after infection with a low input dose (0.001
p.f.u./ceU). The numbers in parentheses indicate the IFN titre.
multiplicity of 10 p.f.u./ceU. Fig. 1 demonstrates that our L cell-NDV system had essentially
the same characteristics as noted above. The data indicate further that the limited replication
is not a general phenomenon observed throughout the whole course of infection. Until about 7
h after infection the multiplication appeared to progress normally and the ratios of infectivity
to haemagglutination (p.f.u. :HA ratio) were comparable to those in the permissive system.
Thereafter, non-infectious haemagglutinin was predominantly produced instead of infectious
virus, resulting in a gradual decrease in p.f.u. "HA ratio. Furthermore, this change of the
replication pattern seemed to be temporally related to the production and accumulation of
IFN in the system. IFN was first detectable at 6 h after infection, the time shortly before the
production of infectious virions ceased, and continued to accumulate thereafter even when
virus production was entirely suppressed. In contrast to L cells, there was no detectable IFN
production by infected BHK-21 cells, and a typical single step growth was observed with a
final titre approx. 10 times higher than in L cells.
Previous reports have emphasized the following two additional features characteristic of the
L cell-NDV system. One of these is that infectious progeny may remain cell-associated
without being released into the culture medium (Thacore & Youngner, 1970). However, even
in the permissive systems the progeny is found to be cell-bound for long periods until the
infected cells show extensive degeneration (Nagai, 1973; Nagai et al., 1976). Thus, the low
efficiency of virus release could not be a phenomenon strictly related to the L cell-NDV
system. The other feature is based on the observation of Hecht & Summers (1974), that
virions isolated from infected L cells by sucrose gradient centrifugation do not form plaques
on monolayers of L cells. It was therefore suggested that L cells might produce virions
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112
Y. NAGAI AND OTHERS
defective in composition and in the biological functions required for the establishment of
infection. However, this also seemed unlikely since the same virus preparation was shown to
have a titre as high as 1.5 x 107 p.f.u, if assayed on another host, chick embryo cells. To
substantiate this we isolated virions from infected L and BHK-21 cells by essentially the same
procedure as employed in the study of Hecht & Summers (1974). The cells infected with a
multiplicity of 10 p.f.u./cell were harvested at 11 h by scraping into PBS, sonicated for 30 s
with an ultrasonic oscillator (Tominaga, type UR, 168 W, 20 kHz) and centrifuged at 950 g
for 20 rain. The resulting supernatant fluids were centrifuged at 24000 rev/min for 90 rain
through linear gradients of sucrose (20 to 50 % in PBS) in a SW27 rotor. To facilitate the
identification of virus-containing regions, purified virions grown in chick embryo were run in
parallel. The presumed virus-containing regions were collected and after adjusting to 80 HA
units/ml they were assayed for infectivity on three different hosts. The virions from L and
BHK-21 cells showed equally high titres (about 107 TCIDso/ml) on chick embryo cells as well
as on BHK-21 cells. In contrast, when assayed on L cells the titres were much lower (about
103 TCIDs0/ml) not only with L cell virions but also with those from BHK-21 cells. These
data indicate that L cells produce a certain amount of highly infectious particles which can be
separated from non-infectious haemagglutinin by sucrose gradient centrifugation, and that
their low titre on L cells would be simply a result of limited growth of the virus in these cells.
Fig. 2 demonstrates that the difference in virus yield between the permissive and
non-permissive systems is much greater if infection is initiated with a lower input dose. There
was a difference of approx. 3 log10 in the maximum virus yield when the cells were infected
with a multiplicity of 0.001 p.f.u./cell. Here again, it was found that L cells were able to
support virus growth comparable to that in BHK-21 cells up to 11 h, when IFN was not y e t
detectable.
Effect of anti-mouse IFN serum on the growth of N D V in L cells
We next investigated whether the neutralization of the endogenously produced IFN might
affect the yield of NDV in L cells. The cells were infected with a multiplicity of 10 or 0.001
p.f.u./cell and various amounts of anti-mouse IFN serum were added to the culture medium at
1 h after infection. The control cultures received corresponding concentrations of normal
rabbit serum. As shown in Table 1, 10% anti-IFN serum was found to completely neutralize
the IFN produced during single cycle growth, and a remarkable enhancement of the virus
yield was observed with an increase in the p.f.u. :HA ratio comparable to that found in the
permissive system (see Fig. 1). Under conditions of multiple cycle growth, complete
neutralization of IFN resulted in a dramatic enhancement of the virus production also with a
high p.f.u.: HA ratio. Thus, the limited growth of NDV in L cells could be circumvented by
neutralizing the IFN with specific antiserum. However, the effect of antiserum was either not
appreciable (single cycle growth) or not distinct (multiple cycle growth) if neutralization was
only partial (Table 1). In the latter case it should be noted that the p.f.u. : HA ratio remained at
a low level, although the virus yield was enhanced by a factor of about 103. Thus, it appeared
that relatively small amounts of IFN might be enough to restrict the virus replication.
Since the antiserum used was prepared by immunizing rabbit with IFN induced in L cells,
we also studied the effect of anti-L cell serum. The antiserum was heated for 30 min at 56 °C
since it was highly toxic unless heated. The virus growth was found to be unaffected by the
antiserum. There was neither enhancement of virus yield nor increase in p.f.u. : HA ratio over
widely varying concentrations of the serum (data not shown).
These results suggest that limited replication of NDV in L cells is a result of interference by
the endogenously produced IFN during the course of infection.
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L i m i t e d N D V growth and interferon
(a)
u
1 2
3 4
5 6 7
(b)
U 1 2
113
3 4 4' 5
5'
6
6'
7
-L
-L
-HN
-HN
-NP
-NP
--M
-M
Fig. 3. The polypeptides synthesized in (a) BHK-21 and (b) L cells infected with NDV and incubated
with normal rabbit serum or anti-IFN serum. The cells were infected at an input multiplicity of 10
p.f.u./cell. BHK-21 cells and one set of L cell cultures were incubated with 10% normal rabbit serum
from 1 h after infection and pulse-labelled in the presence of the serum for 1 h with [35S]methionine at
every hour as indicated by the numbers on the top of the gels (U = uninfected). The infected L cells were
incubated with 10% anti-IFN serum and pulse-labelled similarly at 4, 5 and 6 h after infection as
indicated (4', 5' and 6'). The cells were dissociated, subjected to electrophoresis and processed for
autoradiography as described in Methods. The migration is from top to bottom.
T a b l e 1. Effect of anti-IFN serum on the growth of N D V in L cells*
Multiplicity
of infection
10
0.001
Serum
Anti-IFN
Normal
Anti-IFN
Normal
Concentration
(%)
10
10
3
3
Anti-IFN
Normal
Anti-IFN
Normal
10
10
2
2
Virus yield
Time after
:
~'
infection (h) IFN titre
p.f.u.
HA
12
< 20
2.2 × 107
64
12
480
4.0 × 106
64
12
80
3.6 × 106
96
12
320
2.8 × 106
96
48
48
48
48
< 20
120
120
120
5.6
8.0
2.7
2.4
×
x
×
×
107
103
106
103
256
<2
64
<2
p.f.u. :HA
3.4 x 105
6.3 × 104
3.8 × 104
2.9 x 104
2.2 × 105
4.2 × 104
-
* L cells infected with NDV at different multiplicities were incubated with anti-mouse IFN rabbit serum or
normal rabbit serum. The sera were added to the culture medium 1 h after infection to give the final concentrations designated, and the virus and IFN titres determined at the designated times after infection.
Effect of anti-IFN serum on the synthesis of viral proteins in L cells
T h e s y n t h e s i s o f v i r a l p r o t e i n s in L cells i n f e c t e d w i t h N D V w a s a n a l y s e d in t h e p r e s e n c e or
a b s e n c e o f a n t i - I F N s e r u m . T h e cells w e r e i n f e c t e d w i t h a m u l t i p l i c i t y o f 10 p.f.u./cell a n d
i n c u b a t e d in a m e d i u m c o n t a i n i n g 10 % a n t i - I F N s e r u m o r n o r m a l r a b b i t s e r u m a d d e d a t 1 h
after infection. E v e r y h o u r a f t e r i n f e c t i o n t h e cells w e r e labelled for 1 h w i t h [ 3 5 S ] m e t h i o n i n e
a n d t h e l a b e l l e d p r o t e i n s w e r e a n a l y s e d b y e l e c t r o p h o r e s i s o n p o l y a c r y l a m i d e gels. B H K - 2 1
cells, similarly i n f e c t e d a n d l a b e l l e d in t h e p r e s e n c e o f 10 % n o r m a l r a b b i t s e r u m , w e r e also
e x a m i n e d . T h e r e s u l t s a r e s h o w n in Fig. 3 T h e viral p r o t e i n s N P a n d M w e r e first d e t e c t a b l e in
B H K - 2 1 cells a t 2 h a f t e r i n f e c t i o n a n d t h e r a t e o f s y n t h e s i s i n c r e a s e d p r o g r e s s i v e l y u n t i l 8 h
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Y. N A G A I AND O T H E R S
4'
4
NP
NP
M
5'
"~--NP
6'
.4----NP
NP
M
NP
M
M
Fig. 4. Densitometry scanning of the lanes 4, 4', 5, 5', 6 and 6' of the autoradiogram shown in Fig.
3 (b).
(Fig. 3 a). This was followed by a gradual decrease in the rate of synthesis (not shown). In L
cells incubated with normal serum, viral proteins were also detected at 2 h and their synthesis
seemed to progress normally until about 4 h (Fig. 3 b). However, subsequent protein synthesis
remained at a low level without such amplification as was observed during the progress of
infection in BHK-21 cells. However, when infected L cells were incubated with anti-IFN
serum, distinct enhancement of the viral protein synthesis was observed at the later stage of
infection, showing ultimately, synthesis rates almost comparable to those in the permissive
system (see Fig. 3 a). The difference in protein synthesis between antiserum-treated and
non-treated L cells was more clearly shown by densitometry tracing of the autoradiogram
(Fig. 4).
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Limited ND V growth and interferon
115
Although viral proteins other than NP and M were not well resolved under any conditions
employed here, they were clearly detectable when the cells were more heavily infected with an
input dose of 50 p.f.u./cell. It was also found that there was no amplification of their synthesis
in L cells (not shown). These results indicate that the endogenously produced IFN would
suppress the progress of viral protein synthesis and that the suppression could be prevented by
neutralization of the IFN.
DISCUSSION
The data presented in this report show that administration of a specific antiserum against
IFN is extremely valuable in elucidating the role of endogenous IFN in the progress of virus
multiplication. We demonstrated that the limited growth of NDV in L cells could be a result of
interference by the IFN produced during the course of infection. Thus, the restriction of virus
growth was apparent only late in infection when IFN became detectable and accumulated.
IFN production and its feedback into the system would probably inhibit the progress of viral
protein synthesis and lead to the reduction in virus yield.
Production of non-infectious haemagglutinin was initiated concomitantly with the
appearance of IFN. This result is in agreement with other findings with IFN-treated cells
infected with murine leukaemia virus (Pitha et aL, 1976; Wong et al., 1977; Billiau et aL,
1978) and with VSV (Maheshwari & Friedman, 1980), where not only the production of
infectious progeny was inhibited but also the synthesis of non-infectious particles was
enhanced.
When infection was initiated with reduced input doses, great differences in the final virus
yield were observed between BHK-21 and L cells as well as between antiserum-treated and
non-treated L cells. This indicates that the endogenously produced IFN strongly inhibits the
spread of infection in multiple replication cycles. The IFN produced during the initial few
replication cycles may have induced an antiviral state in the uninfected cells before their
infection by progeny virus.
Use of actinomycin D is an alternative way to elucidate the role of IFN. Youngner & Scott
(1968) showed that the suppression of IFN production by the antibiotic had no influence on
the multiplication of NDV in L cells, suggesting no correlation between IFN and the limited
virus growth. We also employed actinomycin D in our L ceU-NDV system. However, due to
a severe drop in the viability of the cells caused by the antibiotic, the data obtained have been
difficult to interpret. For example, under conditions where IFN production was completely
inhibited withminimum loss of the viability there was still a 60 to 70 % decrease in the cellular
activity, as judged by the capacity of another set of L cell cultures to support the growth of
VSV, whose RNA synthesis is known to be unaffected by the antibiotic (data not shown).
Therefore, although a considerable increase in p.f.u. :HA ratio as well as in NDV yield were
occasionally observed on overcoming such non-specific loss of the cellular viability, the
information available was much more limited and less specific than that obtained by the
antiserum procedure. The data of Youngner & Scott (1968) seem to differ significantly in this
respect, since their actinomycin D-treated L cells were able to support normal growth of
Semliki Forest virus under conditions of complete inhibition of IFN induction by NDV.
Youngner and co-workers have further reported that a mutant, NDVpi, isolated from
persistently infected L cells, could replicate well in L cells although it induced much higher
amounts of IFN than the wild-type, NDV o and suggested that this was additional evidence for
the lack of correlation between IFN production and limited virus growth. Therefore, there are
apparently conflicting conclusions regarding the mechanism of growth restriction of NDV in
L cells and it remains to be elucidated whether such a discrepancy is due to differences in virus
strain and cell line or experimental procedure.
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There have been a number of studies dealing with the L cell-NDV system. Some of these
seemed to be carried out on the assumption that I F N might not be responsible for the limited
virus growth. For example, the synthesis of viral R N A and proteins has been analysed in the
presence of actinomycin D, an inhibitor of I F N production (Hecht & Summers, 1974,
Gresland et al., 1979). Our results make it difficult to interpret these data unambiguously,
until the possible influence of I F N on the virus replication is assessed in each case.
We are indebted to Dr S. Kono for his generosity in providing anti-mouse interferon. We thank S.
Hayashi, C. Kobayashi and F. Yamamoto for excellent technical assistance and M. Iinuma for
continuous support. This work was supported in part by research grants from the Ministry of
Education, Science and Culture and the Ministry of Welfare, Japan.
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(Received 10 December 1980)
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