The liposome-encapsulated dichloromethylene-diphosphonate ([Formula: see text]) is known to be toxic to the cultured macrophages and has selective cytotoxic effects on macrophages in rats and mice, but has minimal adverse effects on the non-phagocytic cells following various administration routes. In this study, the cytotoxicity of [Formula: see text] on chicken macrophages was investigated. Similar to findings related to other mammals, the [Formula: see text] was toxic to the macrophages originating from the chicken peripheral blood mononuclear cells as indicated by the MTT (3-(4, 5-dimethylthiazole-2-yl) 2, 5-diphonyltetrazolium bromide) cleavage assays. After administered with [Formula: see text], chicken spleens were rapidly and progressively reduced in sizes. This reduction was more severely gross than with the experimental course. One day post-treatment, the histopathology showed that most of the macrophages around the peri-ellipsoid white pulp had undergone marked cellular necrosis and lysis, and more severe lesions appeared with the loss of lymphoid and reticuloendothelial tissues at 3 and 5 days post-treatment. White pulp which has been replaced by red pulp in the spleen were noticed at 3 and 5 days post-treatment. The flow cytometric assays further confirmed the depletion of the macrophages in the chicken spleens following the administration of [Formula: see text], when compared with those of the controls. Furthermore, the replication of the avian reovirus (ARV) in the spleens was significantly reduced in the macrophage-depleted chickens administered with [Formula: see text]. Thus, depletion of the macrophages with [Formula: see text] followed by functional assesses in the macrophage-depleted chicken could be used as a model to confirm the involvement of the macrophages in ARV replication.

Circulating monocytes and tissue macrophages were suggested to be susceptible to avian reovirus (ARV) infection. To determine if ARV infects and replicates in mononuclear phagocytes (KUL01-positive cells), we infected 3-day-old specific-pathogen-free chickens with ARV strain 2408 by inoculation of the left footpad. The left footpads and spleens were collected for analysis at 1.5 and 2.5 d after inoculation. Replication of ARV in the footpad and spleen was demonstrated by detection of the viral protein σNS using immunohistochemical testing and viral S1 RNA expression by real-time quantitative polymerase chain reaction (qPCR). Furthermore, immunofluorescent double-staining assay of cytocentrifuged cells and cryosections of the footpad and spleen for the viral protein σNS and the surface marker recognized by monoclonal antibody (MAb) KUL01 indicated that KUL01-positive cells costained with MAb H1E1, which recognizes ARV protein σNS. In addition, more ARV S1 RNA was measured by qPCR in the KUL01-positive cell samples prepared from the footpad or spleen 1.5 d after inoculation compared with non-KUL01-positive cell samples. The amounts of ARV S1 RNA in the spleen were significantly lower (P < 0.05) than the amounts in the footpad 1.5 d after inoculation. The results suggest that ARV infects mononuclear phagocytes and then replicates within these cells before migrating to the spleen, where it infects and replicates in KUL01-positive cells.

This study was undertaken to elucidate the cytokine response in chicken infected with strains of avian reovirus (ARV) S1133 and 2408. The expression levels of cytokine mRNA in the spleen and viral S1 RNA in various tissues at 1.5 and 2.5 days post inoculation (dpi) were examined using real-time quantitative PCR. Among the cytokines examined, the mRNA expression levels of IL-6, IFN-γ, IL-10 and iNOS at 2.5 dpi were significantly upregulated and higher in chickens infected with strain 2408 than in chickens infected with strain S1133, particularly IL-6 and IFN-γ. A significantly higher levels of viral S1 RNA were detected in the examined tissues from chickens infected with strain 2408 than with strain S1133 over the experimental course, among which the foot pad and spleen were more predominant. The highest levels of IL-6 and IFN-γ mRNA expression correlated with the viral S1 RNA levels in the spleen and the marked clinical diseases and gross lesions, suggesting that IL-6 and IFN-γ may play a role in the pathogenesis of ARV infection.

In this study, recombinant fowlpox viruses (rFPV/HN) expressing Newcastle disease virus (NDV) HN protein and rFPV/HN/chIL-18 co-expressing chicken IL-18 (chIL-18) and HN protein have been constructed and characterized. The co-expressed rHN/chIL-18 antigen or rchIL-18, expressed by our previous construct rFPV/chIL-18 and co-administered with NDV rHN, was assessed for its immunostimulatory activities and protection against NDV challenge in 2-week-old chickens. Chickens were vaccinated, intramuscularly, with various amounts of rHN or rHN/chIL-18 mixed with mineral oil. Production of hemagglutination–inhibition (HI) antibody depended on the concentration of the injected rHN or rHN/chIL-18. The lower HI antibody titers were obtained in chickens group rHN/chIL-18/6 and rHN/chIL-18/7, receiving 50 ng rHN/16.5 ng chIL-18 with mineral oil and 20 ng rHN/6.6 ng chIL-18 with mineral oil, respectively, compared to those in chickens rHN/6 and rHN/7, respectively receiving 50 ng and 20 ng rHN with mineral oil alone. However, the same protection rates were obtained from chickens in groups rHN/chIL-18/6 and rHN/6. Chicken groups rHN/chIL-18/7 and rHN/chIL-18/8 showed higher protective achievements than those in groups rHN/7 and rHN/8, respectively. When rchIL-18 was co-injected with 20 ng rHN plus mineral oil, low level of HI antibody titer was produced; whereas, higher level of IFN-γ production and full protection rates were obtained. On the other hand, lower levels of IFN-γ production and lower protection rate (67%) were obtained in chickens injected with the same amount of rHN with mineral oil alone. Similar results were obtained when 10 ng rHN was used. Thus, when the concentration of rHN decreased to 50 ng or less, rchIL-18 reduced HI antibody production. The increase in IFN-γ production suggested that the enhancement of the cell-mediated immunity might confer the protection from NDV challenge, even accompanied with low HI antibody induction.

Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60 ng rHN/8 ng chIL-12 with MO, 30 ng rHN/4 ng chIL-12 with MO or 15 ng rHN/2 ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15 ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge.

A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.

The single chain chicken interleukin-12 (chIL-12)- or mature chIL-18-encoding region was cloned into the nonessential gene F11L of fowlpox virus (FPV) to generate the recombinant (r) FPV/chIL-12 (rFPV/chIL-12) or rFPV/chIL-18 for rchIL-12 or rchIL-18 production. Splenocytes of chickens were cultured with various dilutions of binary-ethylenimine (BEI)-inactivated rFPV/chIL-12 or rFPV/chIL-18-infected cell lysate for 48h for interferon-γ (IFN-γ) determination. It was found that 1:10,000 chIL-12 or 1:10 chIL-18 cell lysate stimulated the highest levels of IFN-γ production. When chickens given BEI-treated 1:10 rchIL-12 or rchIL-18 cell lysate, intraperitoneally, or rFPV (0.2×105 TCID50) by wing-web puncture, the highest level of IFN-γ was detected in sera on day 3 postinoculation (dpi). In the splenocyte culture supernatants, the highest level of IFN-γ was detected at 14dpi or 21dpi in responses to rchIL-12 or rchIL-18, respectively. The results indicated that rchIL-12 or rchIL-18 could induce IFN-γ production both in vitro and in vivo assays, suggesting that both are biologically active and may allow them to be used in the future as the biological adjuvant in the poultry vaccine development, particularly co-administering with vaccine antigens.

The sequences and phylogenetic analyses of the M-class genome segments of 12 avian reovirus strains are described. The S1133 M1 genome segment is 2283 base pairs long, encoding a protein μA consisted of 732 amino acids. Each M2 or M3 genome segment of 12 avian reovirus strains is 2158 or 1996 base pairs long, respectively, encoding a protein μB or μNS consisted of 676 and 635 amino acids, respectively. The S1133 genome segment has the 5′ GCUUUU terminal motif, but each M2 and M3 genome segment displays the 5′ GCUUUUU terminal motif which is common to other known avian reovirus genome segments. The UCAUC 3′-terminal sequences of the M-class genome segments are shared by both avian and mammalian reoviruses. Noncoding regions of both 5′- and 3′-termini of the S1133 M1 genome segment consist of 12 and 72 nucleotides, respectively, those of each M2 genome segment consist of 29 and 98 nucleotides, respectively, and those of each M3 genome segment are 24 and 64 nucleotides, respectively. Analysis of the average degree of the M-class gene and the deduced μ-class protein sequence identities indicated that the M2 genes and the μB proteins have the greatest level of sequence divergence. Computer searches revealed that the μA possesses a sequence motif (NH2-Leu-Ala-Leu-Asp-Pro-Pro-Phe-COOH) (residues 458–464) indicative of N-6 adenine-specific DNA methylase. Examination of the μB amino acid sequences indicated that the cleavage site of μB into μBN and μBC is between positions 42 and 43 near the N-terminus of the protein, and this site is conserved for each protein. During in vitro treatment of virions with trypsin to yield infectious subviral particles, both the N-terminal fragment δ and the C-terminal fragment φ were shown to be generated. The site of trypsin cleavage was identified in the deduced amino acid sequence of μB by determining the amino-terminal sequences of φ proteins: between arginine 582 and glycine 583. The predicted length of δ generated from μBC is very similar to that of δ generated from mammalian reovirus μ1C. Taken together, protein μB is structurally, and probably functionally, similar to its mammalian homolog, μ1. In addition, two regions near the C-terminal and with a propensity to form α-helical coiled-coil structures as previously indicated are observed for each protein μB. Phylogenetic analysis of the M-class genes revealed that the predicted phylograms delineated 3 M1, 5 M2, and 2 M3 lineages, no correlation with serotype or pathotype of the viruses. The results also showed that M2 lineages I–V consist of a mixture of viruses from the M1 and M3 genes of lineages I–III, reflecting frequent reassortment of these genes among virus strains.