Physical, Chemical, and Biological Properties of Chitosan-Coated Alginate Microparticles Loaded with Porcine Interleukin-1ß: A Potential Protein Adjuvant Delivery System.

We previously developed chicken interleukin-1ß (IL-1ß) mutants as single-dose adjuvants that induce protective immunity when co-administered with an avian vaccine. However, livestock such as pigs may require a vaccine adjuvant delivery system that provides long-lasting protection to reduce the need for successive booster doses. Therefore, we developed chitosan-coated alginate microparticles as a carrier for bovine serum albumin (BSA) or porcine IL-1ß (pIL-1ß) and assessed their physical, chemical, and biological properties. Electrospraying of the BSA-loaded alginate microparticles (BSA/ALG MPs) resulted in an encapsulation efficiency of 50%, and those MPs were then coated with chitosan (BSA/ALG/CHI MPs). Optical and scanning electron microscopy, zeta potential analysis, and Fourier transform infrared spectroscopy were used to characterize these MPs. The BSA encapsulation parameters were applied to ALG/CHI MPs loaded with pIL-1ß, which were not cytotoxic to porcine fibroblasts but had enhanced bio-activity over unencapsulated pIL-1ß. The chitosan layer of the BSA/ALG/CHI MPs prevented burst release and facilitated sustained release of pIL-1ß for at least 28 days. In conclusion, BSA/ALG/CHI MPs prepared as a carrier for pIL-1ß may be used as an adjuvant for the formulation of pig vaccines.

Structural Insight into the Contributions of the N-Terminus and Key Active-Site Residues to the Catalytic Efficiency of Glutamine Synthetase 2.

Glutamine synthetase (GS) catalyzes the condensation of ammonia and glutamate, along with ATP, to form glutamine. Despite extensive studies on GSs from eukaryotes and prokaryotes, the roles of the N-terminus and other structural features in catalysis remain unclear. Here we report the decameric structure of Drosophila melanogaster GS 2 (DmGS2). The N-terminal short helices, α1 and α2, constitute a meander region, and form hydrogen bonds with residues 3-5 in the N-terminal loop, which are not present in the GSs of other species. Deletion of α1 or α1-α2 inactivates DmGS2. Notably, the Arg4 in each monomer of one pentamer forms hydrogen bonds with Glu7, and Asp8 in the adjacent monomer of the other pentamer. Replacement of Arg4 with Asp (R4D) abolishes activity. Analytical ultracentrifugation revealed that Arg4 is crucial for oligomerization. Circular dichroism spectra revealed that R4D may alter the secondary structure. We mutated key residues to identify the substrate-binding site. As Glu140 binds glutamate and Glu311 binds ammonia, mutants E140A and E311A have little activity. Conversely, mutant P214A (P contributes to ATP binding) has higher activity than wild-type DmGS2. These findings expand the understanding of the structural and functional features of the N-terminal meander region of DmGS2 and the residues important for catalytic efficiency.

Chicken interleukin-1ß mutants are effective single-dose vaccine adjuvants that enhance mucosal immune response.

The use of cytokines as adjuvants in poultry is promising because they may enhance immune responses to antigens. In this study, we created two mutants, chicken interleukin-1 beta (ChIL-1ß) Q19A and R140A, which exhibited significantly increased in vivo biological activity compared with wild-type ChIL-1ß. The potential mucosal adjuvant activity of the mutants Q19A and R140A was evaluated in chickens through the intranasal coadministration of a single dose of the Newcastle disease virus (NDV) vaccine with Q19A or R140A. Compared with chickens vaccinated with only the NDV vaccine or the NDV vaccine plus wild-type recombinant ChIL-1ß, chickens vaccinated with Q19A or R140A had significantly increased serum hemagglutination-inhibition antibody titers and anti-NDV-specific IgA antibody levels 1 week later, a high amount of interferon-γ secretion from splenocytes, and increased secretory IgA accumulated in nasal tissues. In addition, molecular dynamics simulations of the mutant R140A bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) were more energetically favorable than the analogous wild-type ternary complex resulting in a decreased energy, which may stabilize the R140A/IL-1RI/IL-1RAcP complex. In conclusion, the mutants Q19A and R140A are effective adjuvants that accelerate and enhance chicken mucosal immunity when co-administered with one dose of the NDV vaccine.

Functional characterization of Helicobacter pylori 26695 sedoheptulose 7-phosphate isomerase encoded by hp0857 and its association with lipopolysaccharide biosynthesis and adhesion.

Helicobacter pylori is a notorious human pathogen and the appearance of antibiotic resistance of this bacterium has posed a serious threat to human health. Lipopolysaccharide (LPS) is a key virulence factor and plays important roles in pathogenesis of H. pylori infection. Sedoheptulose 7-phosphate isomerase (GmhA), as an enzyme participating in the first step of heptose biosynthesis, is indispensable for the formation of inner core oligosaccharide of LPS. In this study, we cloned one putative gmhA ortholog, hp0857, from H. pylori 26695 and overexpressed it in Eschericha coli. Based on the results of molecular weight determination, the recombinant HP0857 is likely a homodimer. Analysis of enzymatic kinetic properties of this protein confirmed that hp0857 is indeed encoded a phosphoheptose isomerase which can utilize sedoheptulose 7-phosphate as the substrate in the ADP-L-glycero-D-manno-heptose (ADP- L,D-Hep) biosynthesis pathway. We also generated an HP0857 knockout mutant and explored its phenotypic changes. This mutant exhibited a decreased growth rate and displayed a "deep rough" type of LPS structure. In addition, it also had a slight decrease in its motility and was more susceptible to hydrophobic antibiotic novobiocin and detergents Triton X-100 and SDS. Furthermore, the adhesive capacity of the HP0857 knockout mutant to AGS cells was reduced significantly, and most of the infected cells didn't show a classic hummingbird phenotype. However, complementation of the HP0857 knockout mutation restored most of these phenotypic changes. In conclusion, we demonstrated that HP0857 protein is essential for inner core biosynthesis of H. pylori LPS and is a potential target for developing new antimicrobial agents against H. pylori infection.

Structure and function of chicken interleukin-1 beta mutants: uncoupling of receptor binding and in vivo biological activity.

Receptor-binding and subsequent signal-activation of interleukin-1 beta (IL-1ß) are essential to immune and proinflammatory responses. We mutated 12 residues to identify sites important for biological activity and/or receptor binding. Four of these mutants with mutations in loop 9 (T117A, E118K, E118A, E118R) displayed significantly reduced biological activity. Neither T117A nor E118K mutants substantially affected receptor binding, whereas both mutants lack the IL-1ß signaling in vitro but can antagonize wild-type (WT) IL-1ß. Crystal structures of T117A, E118A, and E118K revealed that the secondary structure or surface charge of loop 9 is dramatically altered compared with that of wild-type chicken IL-1ß. Molecular dynamics simulations of IL-1ß bound to its receptor (IL-1RI) and receptor accessory protein (IL-1RAcP) revealed that loop 9 lies in a pocket that is formed at the IL-1RI/IL-1RAcP interface. This pocket is also observed in the human ternary structure. The conformations of above mutants in loop 9 may disrupt structural packing and therefore the stability in a chicken IL-1ß/IL-1RI/IL-1RAcP signaling complex. We identify the hot spots in IL-1ß that are essential to immune responses and elucidate a mechanism by which IL-1ß activity can be inhibited. These findings should aid in the development of new therapeutics that neutralize IL-1 activity.

Case study of hydrogen bonding in a hydrophobic cavity.

Protein internal hydrogen bonds and hydrophobicity determine protein folding and structure stabilization, and the introduction of a hydrogen bond has been believed to represent a better interaction for consolidating protein structure. We observed an alternative example for chicken IL-1ß. The native IL-1ß contains a hydrogen bond between the Y157 side-chain OηH and I133 backbone CO, whereby the substitution from Tyr to Phe abolishes the connection and the mutant without the hydrogen bond is more stable. An attempt to explain the energetic view of the presence of the hydrogen bond fails when only considering the nearly identical X-ray structures. Here, we resolve the mechanism by monitoring the protein backbone dynamics and interior hydrogen bond network. IL-1ß contains a hydrophobic cavity in the protein interior, and Y157 is one of the surrounding residues. The Y157 OηH group introduces an unfavorable energy in the hydrophobic cavity, therefore sequestering itself by forming a hydrogen bond with the proximate residue I133. The hydrogen bonding confines Y157 orientation but exerts a force to disrupt the hydrogen bond network surrounding the cavity. The effect propagates over the entire protein and reduces the stability, as reflected in the protein backbone dynamics observed by an NMR hydrogen-deuterium (H/D) exchange experiment. We describe the particular case in which a hydrogen bond does not necessarily confer enhanced protein stability while the disruption of hydrophobicity must be integrally considered.

Structural basis for the specific recognition of IL-18 by its alpha receptor.

Interleukin 18 (IL-18), a member of the IL-1 family of cytokines, is an important regulator of innate and acquired immune responses. It signals through its ligand-binding primary receptor IL-18Rα and accessory receptor IL-18Rß. Here we report the crystal structure of IL-18 with the ectodomain of IL-18Rα, which reveals the structural basis for their specific recognition. It confirms that surface charge complementarity determines the ligand-binding specificity of primary receptors in the IL-1 receptor family. We suggest that IL-18 signaling complex adopts an architecture similar to other agonistic cytokines and propose a general ligand-receptor assembly and activation model for the IL-1 family.

Circular permutation of chicken interleukin-1 beta enhances its thermostability.

Interleukin-1ß is a cytokine critically involved in immune and inflammatory responses. To extend its use as a component of avian vaccines, a circularly permuted chicken interleukin-1ß was synthesized that maintains its activity after pre-incubation at high temperatures, unlike wild-type chicken interleukin-1ß, which is irreversibly inactivated at high temperatures.

Proteomics analysis of the DF-1 chicken fibroblasts infected with avian reovirus strain S1133.

BACKGROUND: Avian reovirus (ARV) is a member of the Orthoreovirus genus in the Reoviridae family. It is the etiological agent of several diseases, among which viral arthritis and malabsorption syndrome are the most commercially important, causing considerable economic losses in the poultry industry. Although a small but increasing number of reports have characterized some aspects of ARV infection, global changes in protein expression in ARV-infected host cells have not been examined. The current study used a proteomics approach to obtain a comprehensive view of changes in protein levels in host cells upon infection by ARV. METHODOLOGY AND PRINCIPAL FINDINGS: The proteomics profiles of DF-1 chicken fibroblast cells infected with ARV strain S1133 were analyzed by two-dimensional differential-image gel electrophoresis. The majority of protein expression changes (≥ 1.5 fold, p<0.05) occurred at 72 h post-infection. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 51 proteins with differential expression levels, including 25 that were upregulated during ARV infection and 26 that were downregulated. These proteins were divided into eight groups according to biological function: signal transduction, stress response, RNA processing, the ubiquitin-proteasome pathway, lipid metabolism, carbohydrate metabolism, energy metabolism, and cytoskeleton organization. They were further examined by immunoblotting to validate the observed alterations in protein expression. CONCLUSION/SIGNIFICANCE: This is the first report of a time-course proteomic analysis of ARV-infected host cells. Notably, all identified proteins involved in signal transduction, RNA processing, and the ubiquitin-proteasome pathway were downregulated in infected cells, whereas proteins involved in DNA synthesis, apoptosis, and energy production pathways were upregulated. In addition, other differentially expressed proteins were linked with the cytoskeleton, metabolism, redox regulation, and stress response. These proteomics data provide valuable information about host cell responses to ARV infection and will facilitate further studies of the molecular mechanisms underlying ARV pathogenesis.

Experimentally validated novel inhibitors of Helicobacter pylori phosphopantetheine adenylyltransferase discovered by virtual high-throughput screening.

Helicobacter pylori is a major etiologic agent associated with the development and maintenance of human gastritis. The goal of this study was to develop novel antibiotics against H. pylori, and we thus targeted H. pylori phosphopantetheine adenylyltransferase (HpPPAT). PPAT catalyzes the penultimate step in coenzyme A biosynthesis. Its inactivation effectively prevents bacterial viability, making it an attractive target for antibacterial drug discovery. We employed virtual high-throughput screening and the HpPPAT crystal structure to identify compounds in the PubChem database that might act as inhibitors of HpPPAT. d-amethopterin is a potential inhibitor for blocking HpPPAT activity and suppressing H. pylori viability. Following treatment with d-amethopterin, H. pylori exhibited morphological characteristics associated with cell death. d-amethopterin is a mixed inhibitor of HpPPAT activity; it simultaneously occupies the HpPPAT 4'-phosphopantetheine- and ATP-binding sites. Its binding affinity is in the micromolar range, implying that it is sufficiently potent to serve as a lead compound in subsequent drug development. Characterization of the d-amethopterin and HpPPAT interaction network in a docked model will allow us to initiate rational drug optimization to improve the inhibitory efficacy of d-amethopterin. We anticipate that novel, potent, and selective HpPPAT inhibitors will emerge for the treatment of H. pylori infection.

Substitution of asparagine 76 by a tyrosine residue induces domain swapping in Helicobacter pylori phosphopantetheine adenylyltransferase.

Phosphopantetheine adenylyltransferase (PPAT) catalyses the penultimate step in coenzyme A biosynthesis in bacteria and is therefore a candidate target for antibacterial drug development. We randomly mutated the residues in the Helicobacter pylori PPAT sequence to identify those that govern protein folding and ligand binding, and we describe the crystal structure of one of these mutants (I4V/N76Y) that contains the mutations I4 → V and N76 → Y. Unlike other PPATs, which are homohexamers, I4V/N76Y is a domain-swapped homotetramer. The protomer structure of this mutant is an open conformation in which the 65 C-terminal residues are intertwined with those of a neighbouring protomer. Despite structural differences between wild-type PPAT and IV4/N76Y, they had similar ligand-binding properties. ATP binding to these two proteins was enthalpically driven, whereas that for Escherichia coli PPAT is entropically driven. The structural packing of the subunits may affect the thermal denaturation of wild-type PPAT and I4V/N76Y. Mutations in hinge regions often induce domain swapping, i.e. the spatial exchange of portions of adjacent protomers, but residues 4 and 76 of H. pylori PPAT are not located in or near to the hinge region. However, one or both of these residues is responsible for the large conformational change in the C-terminal region of each protomer. To identify the residue(s) responsible, we constructed the single-site mutant, N76Y, and found a large displacement of α-helix 4, which indicated that its flexibility allowed the domain swap to occur.

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase.

Helicobacter pylori is a bacterium that causes chronic active gastritis and peptic ulcers. Drugs targeting H. pylori phosphopantetheine adenylyltransferase (HpPPAT), which is involved in CoA biosynthesis, may be useful. Herein, we report the expression in Escherichia coli and purification of recombinant HpPPAT and describe a crystal structure for an HpPPAT/CoA complex. As is the case for E. coli PPAT (EcPPAT), HpPPAT is hexameric in solution and as a crystal. Each protomer has a well-packed dinucleotide-binding fold in which CoA binds. Structural characterisation demonstrated that CoA derived from the E. coli expression system bound tightly to HpPPAT, presumably to initiate feedback inhibition. However, the interactions between the active-site residues of HpPPAT and CoA are not identical to those of other PPATs. Finally, CoA binding affects HpPPAT thermal denaturation.

Potentiation of cell-mediated immune responses against recombinant HN protein of Newcastle disease virus by recombinant chicken IL-18.

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 20ng 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.

Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein.

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 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng 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 15ng 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.

Structural and functional comparison of cytokine interleukin-1 beta from chicken and human.

Interleukin-1 beta (IL-1ß) is an important cytokine in the immune system. The properties of avian IL-1ßs are less well understood than the mammalian IL-1ßs, and there is no available structure of avian IL-1ßs in the Protein Data Bank. Here, we report the crystal structures of wild-type and Y157F mutant IL-1ßs from chicken. Both the wild-type and mutant IL-1ßs share a beta-trefoil conformation similar to that of human IL-1ß and also have an internal hydrophobic cavity. However, the cavity sizes clearly differ from that of human IL-1ß due to the packing of hydrophobic residues. Our studies also reveal that the relative thermal stability of IL-1ßs does not correlate with cavity size but rather is dependent on the amino acid residues present around the cavity. This cavity serves as a scaffold for maintaining the structure of the IL-1ß core region but does not have a biological function per se. Moreover, we found that human IL-1ß cannot induce chemokine expression in chicken fibroblasts or elevate plasma cortisol levels in chickens, implying a lack of cross-species bioactivity. Close examination reveals that significant structural and sequence differences occur in the terminal and some loop regions between human and chicken IL-1ßs. These variable regions have been shown to be critical for receptor binding, thus resulting in a lack of species cross-reactivity between human and chicken IL-1ß.

Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers.

Mitochondria are key organelles in mammary cells responsible for several cellular functions including growth, division, and energy metabolism. In this study, mitochondrial proteins were enriched for proteomics analysis with the state-of-the-art two-dimensional differential gel electrophoresis and matrix-assistant laser desorption ionization-time-of-flight mass spectrometry strategy to compare and identify the mitochondrial protein profiling changes between three breast cell lines with different tumorigenicity and metastasis. The proteomics results demonstrate more than 1,500 protein features were resolved from the equal amount pooled from three purified mitochondrial proteins, and 125 differentially expressed spots were identified by their peptide finger print, in which, 33 identified proteins belonged to mitochondrial proteins. Eighteen out of these 33 identified mitochondrial proteins such as SCaMC-1 have not been reported in breast cancer research to our knowledge. Additionally, mitochondrial protein prohibitin has shown to be differentially distributed in mitochondria and in nucleus for normal breast cells and breast cancer cell lines, respectively. To sum up, our approach to identify the mitochondrial proteins in various stages of breast cancer progression and the identified proteins may be further evaluated as potential breast cancer markers in prognosis and therapy.

Comparative analysis of receptor binding by chicken and human interleukin-1ß.

Interleukin-1ß (IL-1ß) is an important cytokine in the immune system. Mammalian and avian IL-1ßs share only 31-35% sequence identity, and the function of avian IL-1ßs is less well understood by comparison. Although chicken and mammalian IL-1ßs have similar tertiary structures, these ILs differ significantly with respect to receptor activation. Analysis of the structures and sequences of IL-1ßs reveals that the major differences lie in loops. Modeling docking of chicken IL-1ß to its receptor reveals that these variable loops are critical for receptor binding. Molecular dynamics simulations of the IL-1ßs reveal significant changes in the dynamic range of motion upon receptor binding. Loops 3 and 9 of the unbound chicken IL-1ß had greater fluctuations compared with the other loops. Upon binding, the flexibility of these loops, which directly contact the receptor, markedly decreases. Taken together, these results suggest that receptor binding leads to not only favorable enthalpy but also lower conformational entropy.

Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens.

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.

Development of ELISA kits for antibodies against avian reovirus using the sigmaC and sigmaB proteins expressed in the methyltropic yeast Pichia pastoris.

Both the sigmaC and sigmaB proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 sigmaC, 1071-1 sigmaC, S1133 sigmaB, and S1133 sigmaC-sigmaB fusion genes of ARV were cloned into a secreted vector pPICZalphaA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV sigmaC, sigmaB, and sigmaC-sigmaB fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1sigmaC and S1133 sigmaB and S1133 sigmaC-sigmaB fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1sigmaC/S1133 sigmaB and S1133 sigmaC-sigmaB ELISA kits were 0.24 and 0.12, respectively. Forty-four serum neutralization test-positive and twenty-eight serum neutralization-negative samples from vaccinated and commercial farm chickens were tested by the new ELISA kits and by the conventional ELISA. The new ELISA kits have higher positive rates than the conventional ELISA. The results revealed that the correlation rates for the serum neutralization titer and the absorbance values with the new ELISA kits and the conventional ELISA were 100% and 95.8%, respectively.

Avian reovirus core protein muA expressed in Escherichia coli possesses both NTPase and RTPase activities.

Analysis of the amino acid sequence of core protein muA of avian reovirus has indicated that it may share similar functions to protein mu2 of mammalian reovirus. Since mu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant muA ( muA) was designed and used to test these activities. muA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that muA possessed NTPase activity that enabled the protein to hydrolyse the beta-gamma phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k(cat) values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of muA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein muA. The activity of muA is dependent on the divalent cations Mg(2+) or Mn(2+), but not Ca(2+) or Zn(2+). Optimal NTPase activity of muA was achieved between pH 5.5 and 6.0. In addition, muA enzymic activity increased with temperature up to 40 degrees C and was almost totally inhibited at temperatures higher than 55 degrees C. Tests of phosphate release from RNA substrates with muA or K408A/K412A muA indicated that muA, but not K408A/K412A muA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of muA might be carried out at the same active site, and that protein muA could play important roles during viral RNA synthesis.