Immune status assessment by abundance of IFN-alpha and IFN-gamma mRNA in chicken blood.

Avian diseases, including such viral infection as infectious bursal disease, infectious anemia, and Marek's disease, often cause immunosuppression, leading to more severe infection, problems with secondary infection, and inadequate responses to vaccination. Immunosuppression thus causes serious economic losses in commercial poultry production. To date, methods for assessing immune status have been too slow to be of practical help. Reasoning that immunosuppression should be reflected by reduced production of interferons (IFN) in response to a viral antigen, we have developed competitive nucleic acid hybridization microtiter plate assays for chicken IFN-alpha (ChIFN-alpha) and ChIFN-gamma mRNA. To evaluate the assay, chickens were challenged with inactivated Newcastle disease virus (iNDV). Whole blood samples were collected at various times subsequently and preserved with a cationic detergent. Later, total RNA was extracted, and mRNA for both ChIFN-alpha and ChIFN-gamma was measured. Both rose from undetectable levels to reach a peak by 4 h, remained high for about 3 days, and fell to undetectable levels by day 5. Results were similar in chickens aged between 1 and 28 days. In later experiments, blood was collected 4 h after viral challenge. When chickens were immunosuppressed by administering 4-5 mg cyclophosphamide (CY) daily for 3 days and challenged with iNDV, they transcribed less ChIFN-alpha and ChIFN-gamma mRNA, and their antibody response was impaired. Our results suggest that suspected immunosuppression in a commercial flock could be assessed within 2-3 days by challenging birds with iNDV and measuring the amounts of ChIFN-alpha and ChIFN-gamma mRNA in blood obtained 2-4 h later.

Combined sequential treatment with interferon and dsRNA abrogates virus resistance to interferon action.

Many viruses have evolved mechanisms to resist the action of interferon (IFN). These include production of viral gene products that sequester double-stranded RNA (dsRNA) and of small helical RNA. These potentially prevent activation of dsRNA-dependent pathways of IFN action or block expression of cellular genes activated exclusively by dsRNA that may contribute to the antiviral state. Thus, dsRNA might be rate limiting in the development of an IFN-mediated antiviral state. In support of this hypothesis, dsRNA added exogenously to IFN-treated cells in the form of poly(rI):poly(rC) is shown to establish in a dose-dependent manner an antiviral state against two viruses otherwise highly refractory to IFN action, avian reovirus (ARV) and Newcaste disease virus (NDV). Cells exposed singly to high doses of IFN or dsRNA reduced the plaque-forming capacity of these viruses on chicken embryo cells 2-fold. When used in combination, there was up to a 100-fold reduction. In order to abrogate IFN resistance, dsRNA must be added after, not before, an IFN-mediated latent antiviral state is established. dsRNA added exogenously is thought to achieve the threshold required for activation of dsRNA-dependent pathways of IFN action or to induce some dsRNA-stimulated gene whose product acts synergistically with that of some IFN-stimulated gene. The combined sequential treatment with IFN and dsRNA may be useful in overcoming the anti-IFN activity of viruses of clinical interest or in other clinical conditions.

Cellular responses in chickens treated with IFN-alpha orally or inoculated with recombinant Marek's disease virus expressing IFN-alpha.

Mammalian type I interferons (IFN-alpha/beta) are potent mediators of innate antiviral immune responses, in particular through enhancement of natural killer (NK) cell cytotoxicity. Recently, chicken IFN-alpha (ChIFN-alpha) has been identified and shown to ameliorate Newcastle disease virus (NDV) infection when given to chickens at relatively high concentrations in the drinking water. In this report, the effect of recombinant ChIFN-alpha (rChIFN-alpha) on NK cell cytotoxicity was examined using (51)Cr-release assays. NK cell cytotoxic activity was also analyzed following inoculation with attenuated Marek's disease virus (MDV) serotype 1 strain R2/23 and a recombinant MDV (parent strain R2/23)-expressing ChIFN-alpha [rMDV(IFN-alpha)]. Treatment of chickens with high doses of rChIFN-alpha in the drinking water significantly decreased NK cell cytotoxicity compared with untreated chickens over a 7-day period. Inoculation of chickens with R2/23 significantly decreased NK cell cytotoxicity as well, whereas the rMDV(IFN-alpha) had no effect on NK cell cytotoxicity. Treatment of chicken embryo cell cultures with rChIFN-alpha inhibited replication of the very virulent MDV RB-1B strain in vitro, and oral treatment of chickens with rChIFN-alpha reduced MDV R2/23 replication in vivo.

Chicken interferon type I inhibits infectious bronchitis virus replication and associated respiratory illness.

Infectious bronchitis virus (IBV) causes an economically important respiratory disease in poultry worldwide. Previous studies have shown that CD8(+) cytotoxic T lymphocytes (CTL) are critical in controlling acute IBV infection, but the role of innate immunity is unknown. This study describes the in vitro and in vivo anti-IBV activity of natural spleen cell-derived and recombinant chicken interferon type I (rChIFN-alpha). Both natural and rChIFN-alpha inhibited replication of the Beaudette strain of IBV in chicken kidney cells (CKC) in a dose-dependent manner, with the antiviral activity of the former accounted for entirely by its content of type I IFN. IFN at 100 U/ml reduced viral replication by 50% as measured by syncytia formation. In addition, the spleen cell-derived supernatants (natural IFN) inhibited tracheal ring ciliostasis mediated by the Gray strain of IBV. Optimal protection against IBV-induced respiratory disease was obtained after intravenous or oral administration of ChIFN given 1 day before virus challenge and each of 5 days thereafter. ChIFN-I protected chicks from clinical illness by delaying the onset of the disease and decreasing the severity of illness, demonstrating its potential as an immune enhancer.

Transient resistance of influenza virus to interferon action attributed to random multiple packaging and activity of NS genes.

Interferon (IFN) action survival curves for an avian influenza virus (AIV) in chicken or quail cells showed that 40-60% of the virions in a stock of virus were highly sensitive to the inhibitory effects of chicken IFN-alpha (ChIFN-alpha), whereas the rest were up to 100 times less sensitive. This greater resistance to IFN was transient, that is, was not a stable characteristic, in that virus stocks grown from plaques that formed in the presence of 50-800 U/ml IFN gave rise to virus populations that contained both sensitive and resistant virions. If AIV was serially passaged several times in the presence of IFN, the proportion of transiently IFN-resistant virus was greater. We propose a model to account for this transient resistance of AIV to IFN action based on the reported inactivation of the dsRNA-dependent protein kinase (PKR) and its activator dsRNA by the NS1 protein of influenza virus and also on the increase in the survival of AIV in IFN-treated cells exposed to 2-aminopurine, a known inhibitor of PKR. We suggest that IFN-resistant AIV is generated from a random packaging event that results in virions that contain two or more copies of RNA segment 8, the gene segment that encodes the NS1 protein of AIV, and that these virions will produce correspondingly elevated levels of NS1. The experimental data fit well to theoretical curves based on this model and constructed from the fraction of virus in the population expected by chance to contain one, two, or three copies of the NS gene when packaging an average of 12 influenza gene segments that include the 8 segments essential for infectivity.

Chicken interferon types I and II enhance synergistically the antiviral state and nitric oxide secretion.

This report shows that chicken type I and type II interferons (IFNs), like their mammalian counterparts, act synergistically such that a mixture of the two has much greater activity than that expected from the separate contribution of each type. The degree of antiviral synergy was measured by virus plaque reduction and cytopathic effect (CPE) inhibition in both primary and secondary chicken embryo cell cultures. Mixtures of the two Ch-IFNs produced antiviral effects 3-10 times greater than that expected from strict additivity of each IFN acting alone. At high concentrations of IFN mixtures there was a qualitative shift to an exponential IFN action does-response curve that revealed synergistic effects greater than 100-fold. Synergy resulted even with mixtures containing less than 1 U/ml of either type of Ch-IFN. The antiviral effects developed more rapidly with mixtures than when type I or II Ch-IFN was used alone. Mixtures of the two types of Ch-IFN synergistically potentiated nitric oxide secretion in cells of the HD11 chicken macrophage line. Molecular mechanisms are cited that may account for synergy between type I and type II IFNs, and speculation is offered on the epidemiologic and therapeutic implications of synergy in vivo.

Interferon induction as a quasispecies marker of vesicular stomatitis virus populations.

The interferon (IFN)-inducing capacity of different isolates of vesicular stomatitis virus (VSV) of the Indiana (IN) and New Jersey (NJ) serotypes were measured to assess the extent of variability of this phenotype. Over 200 preparations of wild-type field isolates, laboratory strains, and plaque-derived subpopulations were examined. Marked heterogeneity was found in the ability of these viruses to induce IFN, covering a 10,000-fold range. A good fit to a normal distribution for the log of the IFN yields suggests a continuum of incremental changes in the viral genome may govern the IFN-inducing capacity of consensus populations derived from independently arising infections. A broad range in the magnitude of these changes, skewed towards inducers of high IFN yields, is consistent with a comparable series of ribonucleotide changes in the VSV genome, a sine qua non of a quasispecies population. Plaque- or vesicle-derived populations displayed standard deviations less than the mean IFN yields, though skewed to higher yielders, whereas populations from field and laboratory samples which differed widely in time and origin of isolation gave standard deviations greater than the means. The plaque isolation of IFN-inducing particles of VSV-IN, normally masked in populations by the predominance of non-IFN-inducing particles that suppress IFN induction, and the isolation of potent wild-type IFN-inducing VSV-IN from cows during an outbreak of vesicular stomatitis in a region that had yielded only virus expressing the non-IFN-inducing phenotype in prior and subsequent years, supports the view that genetic bottlenecks are operative in the natural transmission of this disease.

Interferon modulation of Marek's disease virus genome expression in chicken cell lines.

Lines of chicken lymphoblastoid cells were established from local lesions induced by simultaneous injection of Marek's disease virus and various stimulants of T-cell activation. Lines developed with regular medium had relatively high mean rates of spontaneous expression of viral internal antigen (6.2%). In contrast, lines developed and maintained with conditioned medium generated by mixed-lymphocyte reaction had a 62-fold reduction in the mean rate of viral internal antigen expression (0.1%). The expression rate could be modulated by the removal or re-addition of conditioned medium to the growth medium. Down regulation involved proteins classified as immediate-early (a 14-kDa polypeptide), early (a 38-kDa phosphoprotein), and late (glycoprotein B homologue) antigens, indicating that the block is very early in virus replication. Once initiated in a given cell, replication apparently proceeded unimpeded. Interferon was determined to be largely responsible for the suppressive activity of the conditioned medium, although involvement of other cytokines could not be ruled out. Also, chicken interferon from other sources, including recombinant interferon, was able to similarly suppress viral antigen expression.

Stages of Marek's disease virus latency defined by variable sensitivity to interferon modulation of viral antigen expression.

Cytokines in conditioned medium can suppress expression of viral internal antigens (VIA) in lymphocytes latently infected with Marek's disease virus. In the present study, conditioned media produced by spleen cells stimulated with concanavalin A or by mixed-lymphocyte reaction had significantly greater (P < 0.05) VIA-suppressive activity with lymphocytes harvested from birds at 14 days post infection than with those collected at 7 days. This finding defines two stages during the latent period in which sensitivity of lymphocytes to cytokine modulation of viral expression differs. Suppression involved proteins representing immediate-early, early and late viral antigens. Physico-chemical characterization of the suppressive factor in conditioned medium was consistent with that expected of interferon. Indeed, natural interferon prepared from avian reovirus-exposed chicken embryo cells, and recombinant chicken interferon, both mimicked the activity of conditioned medium and were more suppressive with lymphocytes from the later stage of latency.

Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active.

We have expressed a cDNA for virus-induced chick interferon in Escherichia coli. The product, a 19-kDa protein lacking the signal peptide, was purified to homogeneity from the bacterial inclusion bodies. Proteins in the insoluble fraction of bacterial lysates were dissolved in guanidine hydrochloride and subjected to chromatography on Q-Sepharose and MonoS columns. Purified recombinant chick interferon has a specific antiviral activity of approximately 10(8) IU/mg and is a powerful inducer of the interferon-responsive promoter of the chicken Mx gene. Culture medium of transfected COS cells expressing full-length chick interferon cDNA contained up to 5 x 10(4) IU antiviral activity/ml that could be neutralized by antibodies to purified recombinant chick interferon. The antibodies precipitated proteins of 23-28 kDa from the supernatants of transfected COS cells. Treatment with endoglycosidase F reduced the size of the immunoprecipitated proteins to approximately 20 kDa, demonstrating that chick interferon is a glycoprotein.

Recombinant chicken interferon: a potent antiviral agent that lacks intrinsic macrophage activating factor activity.

Crude preparations of chicken interferon (ChIFN) from various sources contain both antiviral and macrophage-activating factor (MAF) activity. Previous serological data indicated that unlike mammals, birds might express only a single type of IFN in response to viruses and mitogens that exhibits both activities. We have now expressed a complementary DNA for virus-induced ChIFN in transfected COS cells and in Escherichia coli. Purified recombinant ChIFN is a powerful antiviral agent and has high Mx promoter-inducing activity. However, as the sole agent, recombinant ChIFN lacks MAF activity: it does not induce the secretion of nitric oxide in primary monocyte-derived chicken macrophages. A neutralizing antiserum prepared against cloned ChIFN blocks most of the antiviral and Mx promoter-inducing activity present in preparations of natural ChIFN, but does not inhibit the MAF activity. These results demonstrate that chicken cells can be induced to secrete a novel cytokine which probably represents the avian homolog of mammalian IFN-gamma.

Interferon induction: regulation by both virus and cell.

Both the virus and its host cell play significant roles in the regulation of interferon (IFN) induction and production. The virus exerts a regulatory effect through variation in its capacity to deliver an IFN inducer molecule in the form of dsRNA, and by suppressing activation of the IFN gene(s) or their subsequent expression. Regulation of IFN induction by genetically closely related viruses may encompass a 10,000-fold range of IFN yields. Viruses that fail to induce IFN express a dominant phenotype in that they suppress IFN induction in cells otherwise programed to produce IFN. The loss of the IFN induction-suppressing phenotype through non-temperature-sensitive mutations is accompanied by the acquisition of IFN inducibility from an otherwise latent state. The cell regulates production of IFN through genetically programed events as part of the developmental process in embryos, and through physiological changes that occur during incubation in vitro. In the developing chicken embryo, the acquisition of IFN inducibility may span a 1,000-fold range of IFN yields, and appears as a time-dependent process proceeding uninterrupted from in ovo to in vitro culture. Populations of established cell lines contain subpopulations (clones) that can differ 10-fold from the mean IFN yielding capacity of the parent, and may undergo physiological changes upon "aging" in vitro that also span a 10-fold range of IFN yields. During development, and most likely as a result of physiological changes brought on by aging in vitro, IFN regulatory factors themselves are regulated. This report reviews the regulation of IFN induction and production with vesicular stomatitis virus (VSV) as a model virus, and primary chick embryo cells and an established line of mouse L cells as model host cells to define systematically the extent and manner in which the virus and cell regulate IFN induction and production.

Chicken interferon gene: cloning, expression, and analysis.

A gene encoding chicken interferon (ChIFN) was cloned from a cDNA library made from primary chick embryo cells that had been "aged" in vitro so as to produce copious amounts of IFN upon induction. The coding region is predicted to produce a signal peptide of 31 amino acids and a mature protein of 162 amino acids with a molecular weight of 18,957. There are four potential N-glycosylation sites and six cysteine residues. Three disulfide bonds are possible, with two being common to most mammalian type I IFNs. A motif of 10 amino acids surrounding Cys-137 is highly conserved: It shows 80% homology with mammalian type I IFNs, but only 30% with a reported fish IFN. The T-rich 3' UTR displays the canonical element AATAAA required for polyadenylation, and contains six repeats of the octamer CTATTTAT that may be involved in down-regulating translation. Northern blots demonstrate that the accumulation of ChIFN mRNA correlates with induction of ChIFN determined by bioassay. Biologically active protein was synthesized in transfected mouse L cells using mRNA prepared in vitro from the cloned sequence. This activity was neutralized by a monoclonal antibody prepared against purified ChIFN. The ChIFN gene shows sequence identity at the amino acid/nucleotide level with consensus mammalian IFNs as follows: alpha (24/23%), beta (20/24%), omega (23/43%), tau (20/43%), gamma (3/31%), and with flatfish IFN (16/35%). The conserved features of the predicted ChIFN protein and the general similarity of predicted secondary structure suggest a molecule that fits the five alpha-helix three-dimensional topology reported for type I mammalian IFNs.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon induction by viruses. XXII. Vesicular stomatitis virus-Indiana: M-protein and leader RNA do not regulate interferon induction in chicken embryo cells.

Several field isolates, strains, mutants, and revertants of vesicular stomatitis virus (VSV), Indiana (IN) serotype, were studied that differed greatly in their capacity to induce interferon (IFN) in aged chick embryo cells. The predicted M-protein amino acid sequence of a wild-type field isolate that induced > or = 10,000 units/ml IFN in chicken embryo cells was identical to that of a wild-type field isolate that induced < 2 units/ml and of a noninducing wild-type laboratory strain. The 47-base plus-strand leader RNA sequences were the same for five IFN-inducing, and eight noninducing independent isolates of wild-type VSV IN. Our data show that the M-protein and plus-strand leader RNA do not of themselves regulate the induction of IFN in this system. Because the capacity of VSV IN to induce IFN resides in virion-associated elements (Marcus and Sekellick, 1987, J. Interferon Res. 7, 269-284), the differences in IFN yield observed with various isolates must result from changes in other virion components that remain to be determined.

Interferon induction by viruses. XXI. Vesicular stomatitis virus: interferon inducibility as a phylogenetic marker.

Forty-five vesiculovirus isolates were systematically compared for their capacity to induce interferon (IFN) in chick embryo cells under conditions such that the maximum (quantum) yield of IFN per cell and the titer of IFN-inducing particles (IFP) could validly be determined. Twelve isolates of the New Jersey (NJ) serotype of vesicular stomatitis virus (VSV) were good inducers, yielding amounts of IFN that ranged in a continuum from 300 to more than 8,000 units per 10(7) cells. These must reflect genetic differences between the closely related viruses. These differences were not reflected in the nucleotide sequence of the viral 3' leader RNA, for analysis of eight of the NJ isolates showed no correlation with the IFN yields. As found in previous smaller surveys, 28 out of 32 VSV isolates of the Indiana (IN) serotype produced little or no IFN, or even suppressed its induction. However, four exceptional IN strains were isolated during 1984 and 1985 from cattle within a relatively circumscribed geographical area in Costa Rica and Panama; all belonged to Indiana virus, type 1, subtype IV, in the proposed G-protein gene evolutionary tree. This is the first example of an IFN-inducing phenotype serving as a phylogenetic marker.

Development of the interferon system. I. In chicken cells development in ovo continues on time in vitro.

When confluent monolayers of cells derived from chicken embryos of different gestational age were cultured for several days without a medium change, a condition termed in vitro aging, the cells' developed an increased capacity to express the interferon (IFN) system. The capacity to both produce IFN and to respond to its antiviral action were enhanced up to 1000- and 100-fold, respectively. Remarkably, the programmed development of the IFN system in these cells seemed to continue virtually uninterrupted after monodispersion of the cells and seeding at high cell density. Cells prepared from young embryos required more time to develop the IFN system than cells from older embryos with the yield of IFN, and sensitivity to its action, related directly to the total in ovo and in vitro age of the cells in culture. For example, essentially the same yields of IFN were obtained from cell cultures made from 5-d-old embryos "aged" for 10 d in vitro, as were obtained from 10-d-old embryos whose cells were aged in vitro for 5 d. In contrast, inducibility of 2'-5' oligoadenylate synthetase by IFN and the induction of heat shock genes by elevated temperature are not enhanced with in vitro aging. The programmed development of the IFN system that starts in ovo seems to continue on schedule in vitro, making the development of the IFN system in chick embryo cells appear as a time-dependent process.

Alcohols induce interferon in primary chick embryo cells.

Primary chick embryo cells (CEC), cultured in vitro, produce interferon (IFN) following alcohol treatment. Brief exposure of cells to methanol, ethanol, 2-propanol, or butanol resulted in the production of an acid-labile IFN which could be detected in the supernatant medium as early as 60 min after removal of the drug. Following "priming" of the cells by homologous IFN, these four alcohols induced even greater yields of IFN: up to 4,000 units per 10(7) cells of an acid-stable IFN were detected. In contrast, priming of CEC with IFN had no effect on the yield of IFN induced by a standard viral inducer, avian reovirus. Maximal IFN production by ethanol in IFN-primed cells depended on both the priming dose of IFN and length of contact with the cells, as well as the in vitro age of the cell cultures. Complete neutralization by polyclonal anti-chick IFN serum, of both the acid-labile and acid-stable preparations induced by ethanol treatment confirmed that both were chicken IFNs. This report describes the general characteristics of IFN induction in primary chick embryo cells by alcohols, with particular emphasis on ethanol, chemically the simplest IFN inducer reported to date.

Interferon induction by viruses. XVII. Non-temperature-sensitive mutations regulate interferon induction by vesicular stomatitis virus.

Wild-type (wt) strains of vesicular stomatitis virus (VSV) strain Indiana are poor to non-inducers of interferon (IFN) which express IFN induction-suppressing activity. At non-permissive temperatures, temperature-sensitive (ts) mutants of this virus are either like their wt parents, or they are good to excellent inducers of IFN. IFN inducibility and IFN induction-suppressing activity are mutually exclusive phenotypes in VSV-Indiana. With one exception, all Orsay ts mutants derived by A. Flamand (CNRS, Gif-sur-Yvette, France), representing the five complementation groups, were poor to non-inducers of IFN and were also capable of suppressing IFN induction by other viruses. In contrast, all Glasgow ts mutants derived by C. R. Pringle (University of Warwick, Coventry, U.K.) were excellent inducers of IFN. We demonstrate that this difference in acquisition of IFN inducibility relates primarily to the origin of the mutations; spontaneous for Orsay, and mutagen-derived for Glasgow. Tests with newly generated spontaneous and mutagen-derived mutants, and temperature-stable revertants of IFN-inducing ts mutants indicate that IFN inducibility results from non-ts, multiple mutations rarely acquired spontaneously, but generated frequently upon mutagenesis with 5-fluorouracil. The capacity of VSV-Indiana to induce IFN is considered intrinsic to the virus, but is only manifested when the dominant IFN induction-suppressing phenotype is lost through mutagenesis. Thus, non-ts mutations appear to regulate the expression of the IFN induction-suppressing phenotype, and hence the IFN inducibility of VSV-Indiana.