Abundance of IFN-alpha and IFN-gamma mRNA in blood of resistant and susceptible chickens infected with Marek's disease virus (MDV) or vaccinated with turkey herpesvirus; and MDV inhibition of subsequent induction of IFN gene transcription.
ABSTRACT The effects of the very virulent RB-1B strain of Marek's disease virus (MDV) and turkey herpesvirus (HVT), a vaccinal strain, on abundance of IFN mRNA in the blood were investigated. MDV and HVT infection did not change the circulating level of IFN-gamma mRNA 1 and 7 days p.i., but they increased IFN-alpha mRNA levels slightly in genetically susceptible (to tumour development) B(13)/B(13) chickens. The total number of circulating leukocytes was unchanged and increase in message was accompanied by an increase in circulating CD8alpha(+) and MHC Class II(+) cells. On the contrary, both viruses slightly increased IFN-gamma transcripts and decreased IFN-alpha transcripts in genetically resistant B(21)/B(21) chickens. Further, oncogenic MDV was able to block the response to inactivated Newcastle disease virus, a potent inducer of IFN, in both chicken lines. The inhibiting effect on transcription was present for both IFN at days 1 and 7 p.i. in susceptible B(13)/B(13) chickens, but only at day 7 p.i. in resistant B(21)/B(21) chickens. By contrast, non-oncogenic HVT did not interfere with induction of either message at one day p.i. and MDV had a more suppressive effect than HVT on IFN gene transcription 7 days p.i. in B(21)/B(21) chickens. Thus, the strong ability of MDV to block induction of IFN gene transcription detected in the blood as soon as one day after infection in susceptible chickens, as opposed to resistant chickens, not only causes immunosuppression but also may be related to the virus's oncogenicity.
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ABSTRACT: Interferon activity was measured in media from virally infected chicken embryo fibroblasts and Concanavalin A-stimulated splenic lymphocytes using a viral inhibition assay. Both cell types produce interferon activity. A cDNA probe corresponding to a chicken interferon mRNA was used to probe Northern blots of RNA prepared from both cells. A single hybridizing species of 900 bases was detected in virally infected fibroblast RNA, but no hybridizing species was detected in the splenic lymphocytes. These results suggest that the interferon activity produced by lymphocytes is of different molecular origin than the corresponding activity produced by virally infected fibroblasts.Veterinary Immunology and Immunopathology 11/1996; 53(3-4):269-75. · 1.88 Impact Factor
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ABSTRACT: Successful medical use of interferon for chronic viral infections is increasingly dependent on understanding the biologic and molecular mechanisms of the interferon system. Interferon (IFN) is one of the body's natural defenses. Production of IFN is a defensive response to foreign components of microbes, tumors and antigens. This IFN response begins with the production of the IFN proteins (alpha, beta and gamma) which then induce antiviral, antimicrobial, antitumor, and immunomodulatory actions. Thus, the initial production or administration of IFN(s) does not protect directly but instead reacts with specific receptors on cell surfaces to activate cytoplasmic transduction signals that then enter the nucleus to stimulate cellular genes encoding a number of effector proteins which lead to the defensive actions. The known molecular, humoral and cellular mechanisms by which these effector proteins exert their antiviral activities are presented. In addition, the pathogenesis of chronic infections is overviewed in the context of the interferon defenses.Antiviral Research 08/1994; 24(2-3):97-110. · 3.93 Impact Factor
- Poultry Science 03/1982; 61(2):205-11. · 1.52 Impact Factor
Arch Virol (2005) 150: 507–519
Abundance of IFN-α and IFN-γ mRNA in blood
of resistant and susceptible chickens infected
with Marek’s disease virus (MDV) or vaccinated
with turkey herpesvirus; and MDV inhibition
of subsequent induction of IFN gene transcription
P. Qu´ er´ e1, C. Rivas1, K. Ester2, R. Novak2,
and W. L. Ragland2
1Institut National de la RechercheAgronomique, Bioagresseurs,
Sant´ e et Environnement, Nouzilly, France
2Institut Ruder Boˇ skovi´ c, Division of Molecular Medicine,
Received May 25, 2004; accepted September 17, 2004
Published online December 10, 2004 c ? Springer-Verlag 2004
Summary. The effects of the very virulent RB-1B strain of Marek’s disease
virus (MDV) and turkey herpesvirus (HVT), a vaccinal strain, on abundance
of IFN mRNA in the blood were investigated. MDV and HVT infection did
not change the circulating level of IFN-γ mRNA 1 and 7 days p.i., but they
increased IFN-α mRNA levels slightly in genetically susceptible (to tumour de-
velopment) B13/B13chickens. The total number of circulating leukocytes was
CD8α+and MHC Class II+cells. On the contrary, both viruses slightly increased
chickens. Further, oncogenic MDV was able to block the response to inacti-
vated Newcastle disease virus, a potent inducer of IFN, in both chicken lines.
The inhibiting effect on transcription was present for both IFN at days 1 and 7
p.i. in susceptible B13/B13chickens, but only at day 7p.i. in resistant B21/B21
chickens. By contrast, non-oncogenic HVT did not interfere with induction of
either message at one day p.i. and MDV had a more suppressive effect than HVT
on IFN gene transcription 7 days p.i. in B21/B21chickens.Thus, the strong ability
of MDV to block induction of IFN gene transcription detected in the blood as
soon as one day after infection in susceptible chickens, as opposed to resistant
508P. Qu´ er´ e et al.
Marek’s disease virus (MDV) is a herpesvirus (genus Mardivirus, family Herpes-
potential, serotype 2 without oncogenic potential and serotype 3 comprising a
to vaccinate against oncogenic MDV . Genetic resistance linked to the MHC
able to replicate MDV but they as well as vaccinated susceptible chickens do not
usually develop tumours [52, 60]. Nevertheless, genetic and acquired resistance
to disease can be undermined by very virulent strains of MDV .
but are of prime importance to define a prophylactic strategy. Cell-mediated
immunity is essential to protection . Broader specificity of the T cell cyto-
toxic response against MDV has been identified in genetically resistant chickens
compared to genetically susceptible chickens. Specific antiviral cytotoxicity may
thus be an important protective mechanism [39, 43].The innate immune response
is triggered early in genetically resistant chickens during the first phase of viral
replication. The NK cell response is higher in genetically resistant chickens com-
identified the macrophage as an additional important partner for the mechanisms
involved in the control of viral replication in Marek’s disease [22, 23]. Nitric
oxide (NO) produced by macrophages from L-arginine through the activation
of enzyme-inducible NO synthase (iNOS) has been proved to be involved in
non-specific defence mechanisms against MDV [14, 62]. Our recent findings
show that, following the pattern of iNOS expression, early strong expression of
cytokines with strong iNOS-inducing activity such as IFN-γ and CC chemokines
from the MIP family is observed in genetic resistance and resistance acquired
after vaccination . Resistance to MDV would thus appear to be preferentially
linked to the early establishment of cytokine induction characteristic of a Th1
immune response, at least in the spleen.
MDV has immunosuppressive properties. The initial cytolytic phase of viral
replication (defined as 3 to 7 days p.i.) is characterized in vivo by strong but
time-limited inhibition of mitogen-inducible T-lymphocyte proliferation, both in
genetically susceptible and resistant chickens [30, 45] during the first week p.i.,
37]. General immunosuppression is observed thereafter to the last tumoural stage
in Marek’s disease have focused on the spleen as a lymphoid organ replicating
at a high level after MDV inoculation. The aim of the present study was to
analyse whether the early phase of MDV-induced immunosuppression evidenced
by lymphoproliferation also affects systemic cytokine production, specifically
MDV and ChIFN gene transcription509
IFN-α and IFN-γ, both known to be involved in limitation of herpesvirus replica-
blood for an overall assessment of immune status . Comparison was made
an oncogenic strain of MDV and the vaccinal, non-oncogenic HVT strain.
Materials and methods
from the GB1 Athens strain and the Cornell N strain, respectively. They are incompatible in
graft tolerance tests and were selected for the respective B haplotype by serological typing
under specific pathogen-free (SPF) conditions, were used for the experiments at 6–8 weeks
The very virulent strain of MDV RB-1B  was maintained by successive passages in SPF
outbred chickens. HVT vaccine was purchased from Fort-Dodge Sant´ e Animale (France).
One thousand PFU of each virus were inoculated intramuscularly per chicken.
II, i.e. 12 controls, 12 inoculated with RB-1B and 12 inoculated with HVT. Six chickens per
group were bled (0.1ml in 0.5ml Catrimox-14 (Qiagen, USA)) 24h after MDV or HVT
inoculation and each chicken was then inoculated with inactivated Newcastle disease virus
(iNDV) subcutaneously and blood was taken again 5h later. Inactivated NDV (Pestikal®,
Veterina Ltd., Zagreb, Croatia) was inoculated at 200µl per chicken to induce transcription
of IFN genes . NDV antigen is a potent inducer of IFN-α and IFN-γ in chickens  and
the maximum induction of IFN mRNA is observed at 5h post-injection . Levels of IFN-α
and IFN-γ transcription in total blood were measured before and after iNDV stimulation.
The six chickens remaining per group were tested in the same way 168h (7 days) after MDV
or HVT inoculation.
Two additional experiments (experiment III: 18 B13/B13chickens and experiment IV:
18 B21/B21chickens) were performed to measure number of circulating leukocytes and the
were inoculated with RB-1B or HVT or left untreated. Seven days later, blood was taken and
leukocytes were isolated on LSM.
Assay of chicken IFN-α and IFN-γ mRNA
Peripheral blood was preserved in Catrimox-14, total RNA extracted and IFN mRNA as-
sayed by competitive hybridisation in microtitre plates . A biotinylated plasmid probe
was used in experiment I  and a biotinylated probe produced by PCR in experiment
II. This can be done because the specificity of each probe is determined by nucleic acid
complementarity, which was the same for each type of probe. The PCR-produced biotiny-
lated probes were prepared using specific primers; 5?AGAAGACATAACTATTAGAA3?
(forward) and 5?TTAGCAATTGCATCTCCTCT3?(reverse)  for ChIFN-γ, and 5?ATG
510P. Qu´ er´ e et al.
GCTGTGCCTGCAAGCCCA3?(forward) and 5?CTAAGTGCGCGTGTTGCCTGT3?(re-
verse)  for ChIFN-α. Plasmid DNA containing cDNA for each gene (0.3µg/ml) was
used as a template at 2µl/50µl PCR reaction volume. For biotin labeling, biotin-14-dCTP
initial denaturation at 95◦C for 2min, samples were denatured at 95◦C for 30s, annealed
with primers at 60◦C for 30s, and elongated at 72◦C for 40s. After 30 cycles, samples
were elongated at 72◦C for 7min. Probes were purified on Sephadex G-50 (Sigma)  and
as described .
Briefly, denatured capture probe was immobilized in microtitre plate wells in excess of
target nucleic acid sequence in an RNA extract of the sample. Denatured biotinylated probe
was added in excess of the capture probe.Appropriate conditions for annealing and removal
of excess probe by rinsing were employed.Avidin-alkaline phosphatase complex was added,
Absorbance is proportionally reduced by target that binds to the capture probe.
Enumeration of blood leukocytes, CD4+,
CD8+and MHC Class II+cells
Blood leukocytes were obtained after centrifugation of heparinised blood on LSM 1077
(Eurobio, Les Ulis Cedex B, France). Leukocytes were incubated for 1h at 4◦C with a
chicken-specific CD4 or CD8α  or major histocompatibility Class II  monoclonal
antibody (kind gifts of Drs. M. D. Cooper and C. H. Chen, Departments of Medicine,
Pediatrics and Microbiology, University of Alabama, Birmingham, AL and Dr. N. M. Le
Douarin, Coll` ege de France and CNRS, Institut d’Embryologie, Nogent-sur-Marne, France,
Cergy-Pontoise, France) supplemented with 2% fetal bovine serum (Gibco BRL). Controls
were incubated with an unrelated, isotype-matched, monoclonal antibody.After two washes,
the cells were then incubated with a phycoerythrin-labelled polyclonal anti-mouse IgG goat
antibody (Jackson Lab., West Grove, PA, USA). Cells were analysed by flow cytometry
The JMP statistical program (SAS Institute, Inc., Cary, NC) was used. Data were transformed
by subtracting the absorbance measured from an arbitrary number. This was done so that
larger numbers reflected greater abundance. The transformation does not alter statistical
inference. Data were analysed by non-parametric Wilcoxon/Kruskal-Wallis rank sums tests,
and significant differences were accepted at p≤0.05.
Effect of RB-1B and HVT infection
on IFN gene transcription
Abundance of IFN-α transcripts was decreased in the blood of B21/B21resis-
tant chickens by both viruses 1 and 7 days p.i. (Table 1). Abundance of IFN-γ
transcripts was increased by both viruses in B21/B21chickens at 1 day, whereas
at 7 days p.i. only HVT caused an increase, the level not being affected by
MDV and ChIFN gene transcription511
Table 1. Abundance of mRNA for chicken interferon (ChIFN) before and 5h after challenge
and 7 days after infection with RB-1B strain of Marek’s disease or turkey herpesvirus (HVT)
1Chickens were infected with MDV (RB-1B) or with HVT or left uninfected (Controls).
Each treatment group was housed separately. Blood samples (0.1ml) were collected before
and 5h after challenge with iNDV as an IFN inducer. Blood samples were collected from
different birds per group 1 day and 7 days after MDV or HVT inoculation
2Abundance of mRNA was measured by competitive nucleic acid hybridisation in
microtitre plates. Means±SD of [1-absorbance 405nm], no. of observations in parenthesis
3Means for each ChIFN mRNA at each day after infection that have different alphabetic
superscripts (a−d) are different at p≤0.05 by rank sum tests. Means with the same alphabetic
superscripts are not different
MDV. In contrast, both viruses increased abundance of IFN-α transcripts whereas
Inactivated NDV is a potent IFN inducer in the chicken. One day p.i., neither
virus interfered with induction of transcription in response to iNDV for either
IFN in the blood of the resistant B21/B21chickens. Seven days p.i., both viruses
interfered with transcription of both IFN genes, MDV having a greater effect. A
response to iNDV still was observed for IFN-α mRNA in HVT-infected chickens,
but lower than for non-infected chickens.
The effect of infection on induction of IFN gene transcription by iNDV in
the blood was quite different in B13/B13chickens. MDV blocked induction of
transcription for both IFN genes at both 1 and 7 days p.i., whereas HVT did not
interfere with induction of either message at either day.
Effect of RB-1B and HVT infection on blood
Neither virus altered the total number of circulating leukocytes in either strain
of chicken (Table 3). Seven days p.i., RB-1B and HVT infection had no effect