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Journal of Virology, April 2000, p. 3605-3612, Vol. 74, No. 8
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Inhibitory Effects of Nitric Oxide and Gamma Interferon on In
Vitro and In Vivo Replication of Marek's Disease Virus
Zheng
Xing
and
Karel A.
Schat*
Unit of Avian Health, Department of
Microbiology and Immunology, College of Veterinary Medicine,
Cornell University, Ithaca, New York 14853
Received 28 October 1999/Accepted 25 January 2000
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ABSTRACT |
The replication of Marek's disease herpesvirus (MDV) and
herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF)
cultures was inhibited by the addition of
S-nitroso-N-acetylpenicillamine, a nitric oxide
(NO)-generating compound, in a dose-dependent manner. Treatment of CEF
culture, prepared from 11-day-old embryos, with recombinant chicken
gamma interferon (rChIFN-
) and lipopolysaccharide (LPS) resulted in
production of NO which was suppressed by the addition of
NG-monomethyl L-arginine (NMMA), an
inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures
for 72 h prior to treatment with rChIFN- plus LPS was required
for optimal NO production. Significant differences in NO production
were observed in CEF derived from MDV-resistant N2a (major
histocompatibility complex [MHC],
B21B21) and
MDV-susceptible S13 (MHC,
B13B13) and P2a (MHC,
B19B19) chickens.
N2a-derived CEF produced NO earlier and at higher levels than CEF from
the other two lines. The lowest production of NO was detected in
P2a-derived CEF. NO production in chicken splenocyte cultures followed
a similar pattern, with the highest levels of NO produced in cultures
from N2a chickens and the lowest levels produced in cultures from P2a
chickens. Replication of MDV and HVT was significantly inhibited in CEF
cultures treated with rChIFN- plus LPS and producing NO. The
addition of NMMA to CEF treated with rChIFN- plus LPS reduced the
inhibition. MDV infection of chickens treated with
S-methylisothiourea, an inhibitor of iNOS, resulted in
increased virus load compared to nontreated chickens. These results
suggest that NO may play an important role in control of MDV
replication in vivo.
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INTRODUCTION |
Nitric oxide (NO), a free radical
generated by NO synthase (NOS) from L-arginine, is an
important chemical in numerous physiological processes (e.g., as a
neurotransmitter and vasodilator) (3, 37). NO is also
recognized as an important factor in nonspecific immunity with
microbiocidal activities against a broad spectrum of protozoa, fungi,
bacteria, and viruses (2, 14, 18, 23, 33, 38). NO is
produced constitutively in neurons and endothelial cells by nNOS and
eNOS, respectively. Inducible NOS (iNOS) can become expressed in
macrophages as a result of the production of cytokines and bacterial
toxins. The expression of iNOS is essential for the killing of microbes
and functions of NO in the regulation of immune responses. Chicken iNOS
has recently been cloned and sequenced (34). The levels of
expression of iNOS have been linked to specific major
histocompatibility complex (MHC) haplotypes in macrophages of chickens
(22). The role of NO in viral infections in chickens has not
been studied in detail, but several papers suggest that NO may be
important in the pathogenesis of infection with reovirus
(41) and infectious bursal disease virus (26).
Marek's disease (MD) is a herpesvirus-induced, naturally occurring
lymphoproliferative disease of chickens and is characterized by
transformation of mostly CD3+ CD4+
CD8
lymphocytes (49). Three related serotypes
of MD virus (MDV) have been described (45). All oncogenic
strains belong to serotype 1, while naturally nononcogenic strains
(e.g., SB-1 [47]) isolated from chickens are
classified as serotype 2, and related viruses isolated from turkeys
(e.g., herpesvirus of turkeys [HVT] strain FC-126
[59]) are characterized as serotype 3. All MDV strains belong to the subgroup of Alphaherpesvirinae (4).
Most chickens are vaccinated at hatching or at 18 days of embryonation
with FC-126, attenuated serotype 1 strains, or a mixture of the three serotypes (10).
The pathogenesis of MD is divided into three phases (6, 9,
46). The first phase of infection is characterized by a productive-restrictive infection primarily in B lymphocytes, resulting in cell death and temporary immunosuppression. Virus replication shifts
from B cells to activated, CD4+ T cells during the second
part of this phase. During the second phase, a latent infection will be
established in these T cells between 5 and 10 days postinfection (dpi).
The third phase is characterized by reactivation of virus in
susceptible chickens, which may cause a secondary lytic infection
cycle, followed by immunosuppression and subsequent development of
lymphomas. The role of immune responses during the establishment and
maintenance of latency is not fully understood, but several studies
suggest that specific and nonspecific immune responses may play a role. Antigen-specific cytotoxic T lymphocytes (CTL) against several MDV
proteins can be detected starting at 6 dpi (40). Increased levels of gamma interferon (IFN-) mRNA and iNOS mRNA have been reported between 3 to 15 and 6 to 15 dpi, respectively (60). IFN-
and latency maintaining factor, a unidentified cytokine, are
able to maintain latency in splenocytes when cultured for 48 to 72 h (5, 56). Lee et al. (31) demonstrated the
presence of suppressor macrophages at 7 dpi. These cells were thought
to be responsible for the decreased mitogen responsiveness of T cells which had been reported between 5 and 10 dpi with MDV (52). It is plausible that NO produced by these suppressor macrophages is
responsible for the reduced response to mitogens, as had been suggested
by Pertile et al. (41) in the case of reovirus infection in
chickens. Moreover, it is also possible that NO can directly interfere
with MDV replication as has been shown for herpes simplex virus (HSV)
(13). In this paper, the potential effects of NO on MDV
replication were investigated in vitro and in vivo.
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MATERIALS AND METHODS |
Chickens.
Specific-pathogen-free (SPF) chickens and
embryonated eggs were obtained from the departmental SPF
S13 (MHC, B13B13), P2a
(B19B19), and N2a
(B21B21) flocks (57).
S13 and P2a chickens are highly susceptible to the
development of MD tumors, while N2a chickens are resistant. Chicks were
housed in isolation rooms; feed and water were provided ad libitum.
Reagents.
S-Nitroso-N-acetylpenicillamine
(SNAP), an NO-generating compound; S-methylisothiourea
(SMIT), an inhibitor of iNOS activity; L-arginine and
lipopolysaccharide (LPS) (Escherichia coli serotype O55:B5)
were obtained from Sigma Chemical Co. (St. Louis, Mo.). NG-monomethyl L-arginine (NMMA) was
obtained from Calbiochemical, Inc. (San Diego, Calif.). Recombinant
chicken IFN- (rChIFN-) (15) was expressed in E. coli (specific activity, 106 U/mg) and was kindly
provided by John Lowenthal, CSIRO Animal Health, Geelong, Victoria, Australia.
Cell cultures.
Chicken embryo fibroblast (CEF) and chicken
kidney cell (CKC) cultures were prepared from N2a, P2a, and
S13 embryos at 11 days of incubation and from 2- to
3-week-old N2a chicks, respectively, and cultured as described
previously (51). CEF were cultured in M23 (M199 [GIBCO,
Grand Island, N.Y.]) containing 10% tryptose phosphate, 3% fetal
bovine serum (FBS), 0.65% sodium bicarbonate, and antibiotics.
Maintenance medium (M20.25) contained 0.25% FBS, and phenol red was
omitted from the maintenance medium when NO was measured.
Spleen cell suspensions were prepared from 3-week-old P2a, N2a, and
S13 chickens as described previously (40) and
cultured in RPMI 1640 supplemented with 10% FBS to measure NO
production (experiment 4) or inoculated onto CKC cultures for virus
isolation (experiment 5).
Viruses.
The oncogenic JM16 strain of MDV (48)
was propagated in CKC and used at passage 19 (p19 [JM16/p19]).
Attenuated JM16/p48 (48) and HVT-4 (8), a clone
derived from FC-126 (59), were propagated in CEF.
Nitrite determination.
Nitrite, which is produced from NO in
the presence of H2O and O2, accumulates in
culture medium and reflects the amount of NO production. The
concentration of nitrite was determined by mixing 100 µl of culture
medium with 100 µl of Griess reagent (1% sulfanilamide, 2.5%
phosphoric acid, 0.1% naphthylethylene diamine) in 96-well microtiter
plates (35). The color development was measured at
A550 with a spectrometer (Bio-Tek Instruments, Winooski, Vt.). The concentration of nitrite in the medium was calculated by using a standard curve generated by mixing 0 to 250 µM
sodium nitrite solutions with Griess reagent. Standard curves are
typically linear between 0 and 200 µM nitrite. All experiments were
done in triplicate.
Effects of NO and IFN- on MDV replication in cell
culture.
To measure the production of NO in CEF and the effect on
virus replication, cultures were treated with 0, 200, and 400 µM SNAP
(experiment 1, trials 1 and 2), 50 U of rChIFN- per ml, and/or 25 ng
of LPS per ml after a change of medium to M20.25 (experiment 2, trials
1 to 3). The concentrations of rChIFN- and LPS were based on
preliminary experiments (Z. Xing, unpublished data). In experiment 2, trial 4, the effects of different concentrations of rChIFN- on MDV
replication were investigated. The effects of aging of CEF and MHC
background of the CEF on NO induction and subsequent MDV replication
were investigated by treatment with rChIFN- and/or LPS at 24, 48, and 72 h after seeding (experiment 3, trials 1 to 3). In all
experiments, except experiment 1, trial 1, and experiment 3, trial 1, cell cultures were infected in triplicate with 100 to 200 PFU of
JM16/p48 or HVT at 18 h posttreatment. MDV foci were counted
72 h postinfection. The antiviral effects of the treatments were
expressed as the percent reduction in the number of PFU in treated
cultures compared to the number of PFU in control cultures infected
with MDV. The effects of aging and MHC on the production of NO by
cultured splenocytes were investigated after treatment with 50 U of
rChIFN- per ml and/or 25 ng of LPS per ml (experiment 4) as
described for CEF.
Effect of iNOS inhibition on MDV replication in chickens.
Two trials were conducted in which N2a chickens were treated with SMIT
at 25 (experiment 5, trials 1 and 2) and 50 (experiment 5, trial 2)
mg/kg of body weight every other day starting at 1 day of age. Chickens
were infected at 1 day of age with 1,000 PFU of JM16/p19 and were
sacrificed at 3, 6, 9, 12, and 15 dpi in trial 1 and at 6 dpi in trial
2. Spleens were harvested and splenocytes were prepared from pools of
three spleens as previously described (40). For virus
isolation, 5 × 106 splenocytes were plated on CKC
cultures, and foci were counted after 5 days.
Statistical analysis.
Results are presented as means ± standard error (SE). The SE was multiplied by an index, which was
determined by the degree of freedom for 95% confidence. Statistical
significance at P < 0.05 was determined by either
t test or rank analysis (12, 54).
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RESULTS |
Effects of SNAP on NO production and MDV replication in CEF
(experiment 1).
The addition of 200 or 400 µM SNAP to CEF
resulted in the production of NO in a dose-dependent manner (Fig.
1, panels A, C, E, and G). Production of
NO in CEF was less than 2 µM in the absence of SNAP, but increased up
to 28 µM when 400 µM was added. Treatment with SNAP reduced
replication of JM16 (trial 1) (Fig. 1, panels B and D) and HVT (trial
2) in a dose-dependent manner (Fig. 1, panels F and H).
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FIG. 1.
Production of NO in chicken embryo fibroblast cultures
treated with 200 or 400 µM SNAP (A, C, E, and G) and the effect on
replication of MDV strain JM16 (B and D) and HVT (F and H). Cultures
were inoculated with 100 (B and F) or 200 PFU (D and H) of JM16/p48 or
HVT. The reduction of virus replication in the presence of SNAP is
expressed as the percentage of the number of PFU in nontreated
cultures.
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NO production and inhibition of MDV replication in CEF treated with
rChIFN- plus LPS (experiment 2).
In trial 1, N2a-derived CEF
cultures started to produce NO as early as 6 h after treatment of
48-h cultures with 50 U of rChIFN- per ml and 25 ng of LPS per ml
and increased up to 60 µM at 48 h (Fig.
2, lane 3). In contrast, treatment with
rChIFN- did not induce NO production (Fig. 2, lane 1), while LPS
treatment alone resulted in approximately 20 µM NO (Fig. 2, lane 2).
The production of NO was blocked by the addition of 250 µM NMMA (Fig.
2, lane 4). The inhibition was partly reversed by the addition of 1,000 µM L-arginine to the culture media (Fig. 2, lane 5).
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FIG. 2.
Production of NO in CEF cultures treated with rChIFN-
and/or LPS. Triplicate 48-h-old CEF cultures were treated with 50 U of
rChIFN- per ml and/or 25 ng of LPS per ml, and NO concentrations in
the supernatant fluids were analyzed after 48 h. Treatments: 1, 50 U of rChIFN- per ml; 2, 25 ng of LPS per ml; 3, 50 U of rChIFN-
per ml and 25 ng of LPS per ml; 4, same as treatment 3 plus 250 µM
NMMA; 5, same as treatment 4 plus 1,000 µM L-arginine; 6, control.
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In trials 2 and 3, CEF were treated as before and infected with 200 PFU
of JM16/p48 (trial 2) or HVT (trial 3) per culture
18 h
afterwards, and foci were counted 3 dpi. NO concentrations
were
measured at the time of infection and when foci were counted
at 3 and 6 days postplating, respectively (Fig.
3A and
C). Treatment
with rChIFN-
plus LPS
reduced the number of plaques of JM16 and
HVT by 70 to 75% relative to
the untreated cultures (lanes 3 in
Fig.
3B and D, respectively).
Reduction in the number of PFU was
not observed in CEF treated with LPS
alone (lanes 2 in Fig.
3B
and D, respectively), while treatment with
rChIFN-
alone caused
a nonsignificant reduction in the number of PFU
(lanes 1 in Fig.
3B and D, respectively). Addition of NMMA, blocking
the production
of NO, reversed the inhibition of MDV replication (lanes
4 in
Fig.
3B and D, respectively). However, in this set of experiments,
the addition of
L-arginine did not reverse the effects of
NMMA-induced
blocking of NO production, and accordingly, replication of
JM16
and HVT was not inhibited.
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FIG. 3.
Inhibition of replication of MDV strain JM16/p48 and HVT
in CEF cultures from N2a chickens treated with rChIFN- and/or LPS.
Treatments: 1, 50 U of rChIFN- per ml; 2, 25 ng of LPS per ml; 3, 50 U of rChIFN- per ml and 25 ng of LPS per ml; 4, same as treatment 3 plus 250 µM NMMA; 5, same as treatment 4 plus 1,000 µM
L-arginine; 6, control. The concentrations of NO in the
supernatant fluids were measured at 1 (open bars) and 4 (solid bars)
days after the cultures were treated (A and C). CEF were infected
18 h after the treatments with 200 PFU/culture of JM16 (B) or HVT
(D). The effect of treatment on virus replication is expressed as the
percentage of the number of PFU in nontreated cultures. The
concentrations of NO and percentage reduction in PFU represent the
average values of triplicate cultures.
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To determine the efficacy of the inhibitory effect by rChIFN-
alone, a separate experiment was designed. CEF cultures from
the
N2a line were treated with 1, 4, 16, 64, 128, and 256 U of
rChIFN-
per ml 24 h postseeding and infected with JM16/p48 at
200 PFU/plate. Only treatment with 256 U of rChIFN-
per ml significantly
reduced MDV replication (
P < 0.05). However, 10 µM
NO was detected
after treatment with 256 U/ml, which was significantly
higher
than NO levels in CEF cultures treated with 0 to 64 U of
rChIFN-
per ml (
P < 0.05) (data not
shown).
Effect of aging and MHC background of CEF on NO production and MDV
replication (experiment 3).
It was noticed in preliminary
experiments that the levels of NO production were not always
reproducible, especially when CEF cultures were used soon after
seeding. To determine whether aging of CEF cultures is necessary for
reproducible induction of NO, a comparative study was carried out with
CEF prepared from 11-day-old N2a and P2a embryos (trial 1) (Fig. 4A and
B, respectively). CEF were treated with
rChIFN- and LPS at 24 and 72 h after seeding. Treatment at
72 h resulted in significant levels of NO production in N2a, while
treatment at 24 h failed to induce the production of NO (Fig. 4A,
lane 3). Similar responses were obtained with the CEF from P2a embryos
(Fig. 4B, lane 3), but the levels of NO production were much higher in
N2a- than in P2a-derived CEF, suggesting a possible genetic influence
on NO production. This was further investigated by using CEF cultures
from N2a, S13, and P2a embryos which were prepared at the
same time (trial 2). Cultures were treated with rChIFN- and/or LPS
at 24, 48, and 72 h after seeding, and NO concentrations were
measured 24 h after treatment. The data are summarized in Table
1. Stimulation of 24-h cultures did not
produce NO independently of the origin of the CEF. Significant
differences in NO production were found when CEF were treated at 48 and
72 h. At 48 h, the N2a-derived CEF produced 23.5 µM NO,
while the production was minimal in the P2a- and S13-derived CEF. The
production of NO was markedly increased in CEF from these two lines
when stimulated at 72 h, but the values were still significantly
lower than those in CEF from the N2a line (P < 0.05).
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FIG. 4.
Effect of aging of CEF cultures on NO production induced
by treatment with rChIFN- and/or LPS. CEF were prepared from
11-day-old embryos obtained from N2a (A) and P2a (B) chickens and
plated at 3 × 106 cells per 35-mm-diameter culture
dish. Triplicate cultures were treated 24 (solid bars) and 72 (open
bars) h after plating with 50 U of rChIFN- per ml (treatment 1), 25 ng of LPS per ml (treatment 2), 50 U of rChIFN- per ml and 25 ng of
LPS per ml (treatment 3), the same treatment as treatment 3 plus 250 µM NMMA (treatment 4), the same treatment as treatment 4 plus 1,000 µM L-arginine (treatment 5), or control (treatment 6).
The concentrations of NO were measured 24 h after treatment.
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The effects of aging and NO production on the replication of JM16/p48
were investigated in experiment 4 (Table
2). Replication
was not significantly
inhibited in 24-h-old CEF treated with rChIFN-
plus LPS independent
of the MHC background of the CEF. MDV replication
was significantly
decreased when CEF from N2a embryos were treated
at 48 h. The
addition of 250 µM NMMA abolished the reduction,
but the addition of
arginine eliminated the effect of NMMA. In
48-h-old CEF treated with
rChIFN-
plus LPS, the numbers of PFU
were reduced to 12%
(
P < 0.05) in N2a cultures, but only to 51%
(
P < 0.05) in S
13 and 46% (
P > 0.05) in P2a cultures, compared
to those in untreated cultures,
respectively. The differences
in inhibition of virus replication
between N2a and S
13 and between
N2a and P2a cultures are
significant (
P < 0.05). MDV replication
was also
inhibited in 48-h-old cultures treated with rChIFN-
alone in N2a
(63%), S
13 (56%), and P2a (62%) cultures, but these
reductions were not statistically different from the values in
untreated cultures (
P > 0.05). The inhibition of MDV
replication
after treatment of N2a cultures at 48 h with
rChIFN-
plus LPS
(number of PFU reduced to 12%) was significantly
different (
P < 0.05) from that by rChIFN-
alone
(number of PFU reduced to
63%). The effect of NO on MDV replication
was reversed by the
addition of NMMA. In contrast, there were no
significant differences
in the inhibition of MDV replication in
48-h-old S
13 (
P > 0.05)
and P2a
(
P > 0.05) cultures treated with rChIFN-
or
rChIFN-
plus LPS (Table
2). Treatment of 72-h-old CEF with
rChIFN-
plus
LPS resulted in the production of NO (Table
1) in all
cultures,
and the numbers of PFU were significantly (
P < 0.05) reduced independently
of the MHC background of the CEF
(Table
2). Treatment with rChIFN-
also reduced the replication of
MDV, but these reductions were
not significantly different from the
number of PFU in untreated
cultures. The results of treatments with
rChIFN-
versus those
with rChIFN-
plus LPS were significantly
different (
P < 0.05)
(Table
2). The reduction in MDV
replication was blocked by the
addition of NMMA in N2a and P2a
cultures.
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TABLE 2.
Inhibition of MDV in CEF cultures from three MHC-defined
chicken lines and treated with rChIFN-, LPS, NMMA,
and L-argininea
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NO induction in cultured splenocytes.
To determine if
splenocytes prepared from three-week-old N2a, S13 and P2a
chickens produced different levels of NO, cells were cultured in LM10
medium and treated with 50 U of rChIFN- per ml and/or 25 ng of LPS
per ml. NO was produced only after the combined treatment. The addition
of 250 µM NMMA reduced the NO production, but this was reversed by
the addition of 1,000 µM L-arginine. Splenocytes from N2a
and S13 chickens produced significantly higher levels of NO
than splenocytes from P2a chickens (Table
3).
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TABLE 3.
Production of NO in splenocyte cultures from three
MHC-defined chicken lines treated with rChIFN-, LPS, NMMA,
and L-arginine
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Effect of inhibition of iNOS activity on MDV replication in
vivo.
Treatment of N2a chicks with 25 mg of SMIT per kg every
other day starting at 1 day of age resulted in significantly increased rates (P < 0.05) of virus isolation from 3 to 9 dpi
(Fig. 5A) compared to those of control
infected chicks. At 12 and 15 dpi, the virus isolation rates were still
higher in the SMIT-treated chicks than in the control group, but the
differences were no longer statistically significant (experiment 5, trial 1). In trial 2, N2a chicks were treated with 0, 25, and 50 mg of
SMIT per kg every other day and sacrificed at 6 dpi. Virus isolation
rates were significantly higher in chickens treated with 25 and 50 mg of SMIT per kg than in the control group (P < 0.05),
confirming the data observed in trial 1. There was no significant
difference in virus isolation rates between 25- and 50-mg/kg treatments
with SMIT (Fig. 5B).
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FIG. 5.
Inhibition of MDV replication in vivo by NO. N2a
chickens were injected intramuscularly with 25 mg of SMIT per kg in
trial 1 (A) or 25 and 50 mg/kg in trial 2 (B) every other day starting
at 1 day of age until the birds were euthanized. Spleens were harvested
on 3, 6, 9, 12, and 15 dpi in trial 1 and on 6 dpi in trial 2. Virus
was isolated by inoculating CKC with 5 × 106
splenocytes from pools of two to three spleens. The numbers of PFU
isolated from pools of SMIT-treated chickens and control chickens are
indicated by solid and open circles, respectively. Significant
differences (P < 0.05) between groups are indicated by
an asterisk.
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DISCUSSION |
NO is increasingly being recognized as an important component of
the host's defense against infection (2, 14, 18, 23, 33,
38). The replication of a broad range of DNA and RNA viruses is
inhibited by NO, including several herpesviruses, e.g., HSV-1 (13,
25), Epstein-Barr virus (36), and murine
cytomegalovirus (55). In this report, we provide evidence
that NO inhibits also the replication of MDV in vitro and in vivo.
These results are important for the understanding of the pathogenesis
of MD and also provide an explanation for the importance of macrophages in the pathogenesis of MD, as reported more than 20 years ago (19-21, 27, 42). Two important consequences are the
potential role of NO in suppressing lymphocyte proliferation
(1), which may be important during the early pathogenesis of
MDV (8, 30, 31, 52), and restricting viral replication.
Suppressor macrophages have been reported to reduce mitogen
responsiveness early during infection with MDV (31), which
was considered to be evidence for MDV-induced immunosuppression.
However, reduced mitogen responsiveness was also noted after infection with HVT (30) and SB-1, a serotype 2 nononcogenic MDV strain (52). It is likely that these effects were caused by the
activation of macrophages to induce iNOS. Activation of macrophages
causes increased IFN- production, which in turn can stimulate NO
production. MDV infection has been shown to increase IFN- expression
in splenocytes as early as 3 dpi, and the presence of iNOS could be
demonstrated at 6 dpi (60). The suppressive effects of
macrophage-derived NO on T-cell mitogenic response have been
demonstrated by others in rats (17) and chickens
(41). Suppression of T-cell proliferation around 6 dpi by
MDV has important consequences for the pathogenesis of MD. At that
time, MDV switches from its primary target cell, the B lymphocyte, to
activated T cells in which latent infection is established (6,
46). Restriction of T-cell activation and proliferation by NO
will reduce the supply of activated T cells and reduce the level of
virus-infected cells. Thus, an earlier and/or more pronounced
production of NO in genetically resistant strains may contribute to
genetic resistance by limiting the availability of target cells. In
support of this hypothesis, Calnek et al. (7) reported that
splenocytes of N2a line chickens had a significant lower response to
concanavalin A than splenocytes from P2a chickens.
In vivo inhibition of NO production with SMIT enhanced the level of
virus replication significantly between 3 and 9 dpi (Fig. 5A and B).
These results are comparable with data reported by others in which
chickens were treated with antimacrophage serum, repeated silica
injections, or carrageenan injections to suppress macrophage functions.
These treatments resulted in significantly elevated virus titers and
increased tumor development (19, 20, 27, 29-31).
It will be important to determine if treatment with SMIT during early
pathogenesis or continued treatment afterwards will enhance the
development of tumors in both genetically resistant and susceptible
chicken lines. It will also be interesting to determine if treatment
with SMIT increases the response to concanavalin A in the resistant N2a
chickens. Genetic resistance to MD is linked, in part, to the MHC
haplotype of the host (50), but the mechanism of MHC-based
resistance has not been elucidated. Omar and Schat (40)
reported that CTL responsive to the immediate-early gene ICP4 are
generated in resistant N2a chickens, but not in susceptible P2a
chickens, suggesting that these CTL may contribute to genetic resistance. However, it is also possible that the degree of iNOS induction and subsequent NO production by macrophages plays a role.
Hussain and Qureshi (22) reported the differential iNOS gene
expression in macrophages obtained from Cornell K strain (B15B15), GB1
(B13B13), and GB2
(B6B6) chickens. Interestingly, the
highest expression of iNOS was found in macrophages from the K strain,
which is relatively resistant to MD, and expression was lower in
macrophages from GB1 and GB2, which are more susceptible to MD. The
data in this paper confirm that NO production by splenocytes (Table 3)
may be related to the MHC haplotype with the highest level in N2a
splenocytes and the lowest level in P2a splenocytes. The N and P lines,
derived from a common genetic background, were originally developed by Cole (11) by selection for MD resistance. The N2a and P2a
lines were derived when the F2 generation of an N×P cross
was separated based on MHC expression (57). These lines are
not congenic, and genes other than those coding for the MHC may also
influence the levels of iNOS expression. The response of the
splenocytes from the MD-susceptible S13 line was comparable
to the response of N2a. It has been suggested that the susceptibility
of the S13 line is due to a genetic defectiveness in immune
responsiveness (J. Kaufman, personal communication).
The inhibition of MDV replication in CEF after stimulation by
rChIFN- plus LPS is most likely caused by NO and not by other factors that may be activated by the treatment. First of all, the use
of SNAP, producing NO by a chemical reaction, inhibited MDV and HVT
replication in a dose-dependent manner. Second, the use of NMMA, a
competitive inhibitor of iNOS, reduced NO production in stimulated
cultures, and consequently the inhibition of MDV replication was
reversed. The reduction in NO production was partially reversed by the
addition of the iNOS substrate L-arginine, indicating the
fidelity of the NO inhibition of MDV replication. The fact that the
inhibitor did not completely reverse the antiviral state in CEF may
indicate that iNOS activity is not completely inhibited or that NO is
not the only antiviral component induced after treatment with IFN-
and LPS. This treatment may also result in the production of other
antiviral inhibitors, including tumor necrosis factor alpha,
2'5'-oligoadenylate synthetase, Mx proteins, or the P1/eIF-2 protein
kinase (44).
The observation that CEF can be used to study the effects of NO
production on MDV replication provides an important in vitro model,
especially in view of the observed genetic differences in the levels of
NO production in the different genetic strains. The differences in NO
production obtained by using CEF (Table 1) paralleled to an important
degree the differences in splenocytes (Table 3). It may therefore be
possible to use CEF from pedigreed birds to select for increased NO
production and perhaps improved resistance to MD. Monocyte precursors
are present in 10-day-old embryos (24) and are probably the
major source for the iNOS in CEF cultures (60). The effect
of aging on NO production in response to stimulation with rChIFN-
plus LPS is likely caused by the maturation of the monocyte precursors
in cell culture. A similar observation has been made for the production
of IFN- in CEF, which also increases during aging of the cultures
(53).
The mechanism by which NO inhibits viral replication remains unknown.
Conceivably, NO could exert an antiviral effect through its influence
on host cells in which viral replication occurs. NO is capable of
damaging DNA (16, 39, 58), and viral DNA may be damaged
directly. Herpesviruses produce viral ribonucleotide reductase, an
enzyme that can be inhibited by NO (28, 32). This enzyme
could therefore be the target, although it is not essential for
herpesvirus replication. NO may also bind to metal ions in viral
proteins that are essential for replication. For example, a nitroso
compound reduced human immunodeficiency virus infectivity by binding
and removing zinc from a transcription factor with a zinc finger motif
(43). NO may also affect the virus life cycle by influencing
intracellular signaling pathways regulated by the oxidation of
sulfhydryl groups (36). The exact mechanism by which NO
inhibits MDV replication in vitro and in vivo remains to be determined.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the Cooperative State
Research, Education, and Extension Service, U.S. Department of
Agriculture, under agreement no. 98-35204-6425 and 95-37204-2237 and
USDA Regional Research NE-60.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unit of Avian
Health, Department of Microbiology and Immunology, College of
Veterinary Medicine, Cornell University, Ithaca, NY 14853. Phone: (607)
253-4032. Fax: (607) 253-3384. E-mail: kas24{at}cornell.edu.
Present address: Department of Molecular and Cellular Engineering,
The Institute for Human Gene Therapy, University of Pennsylvania School
of Medicine, Philadelphia, PA 19104.
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Top
Abstract
Introduction
