Identification of Novel Linear Epitopes Located in the Infectious Bronchitis Virus Spike S2 Region.

We identified novel linear epitopes on the infectious bronchitis virus (IBV) spike S2 region. The conformational structure of the IBV spike protein was predicted from a homologous protein, human coronavirus NL63 spike. Although the obtained structure was incomplete, most of the IBV spike protein structure was predicted; the N-terminus of the S1 region could not be predicted due to its variability. In the model, the region located in the proximity of the fusion peptide appeared to be well conserved, and we evaluated the antigenicity of these domains, which are involved in the membrane fusion machinery. Western blotting revealed that IBV TM86 spike residues 686-723 were antigenic. Epitope mapping analysis using synthesized peptides revealed that IBV TM86 spike 669-685 (SNFSTGAFNISLLLTPP), 686-697 (SNPRGRSFIEDL), and 692-703 (SFIEDLLFTSVE) residues were major linear epitopes; two identified epitopes (686-697 and 692-703) were covered by the fusion peptide, and the other epitope (669-685) was adjacent to the fusion peptide. Although the identified epitopes are identically located as the neutralizing epitope in severe acute respiratory syndrome coronavirus, the recombinant protein that includes those epitopes could not elicit neutralizing antibodies against IBV. This is the first report describing IBV spike S2 epitopes located in the proximity of the fusion peptide, and it is suggested that the spike fusion machinery of IBV may differ from that of severe acute respiratory syndrome coronavirus, or, alternatively, IBV may have another mechanism to penetrate the cell membrane.

Turkey herpesvirus with an insertion in the UL3-4 region displays an appropriate balance between growth activity and antibody-eliciting capacity and is suitable for the establishment of a recombinant vaccine.

We constructed turkey herpesvirus (HVT) vector vaccines in which the VP2 gene of infectious bursal disease virus (IBDV) was inserted into the HVT genome in the following regions: UL3-4, UL22-23, UL45-46, and US10-SORF3. We then evaluated the relationship between the gene insertion site and the capacity of the virus to elicit antibodies. rHVT/IBD (US10) showed good growth activity in vitro, with growth comparable to that of the parent HVT. On the other hand, rHVT/IBD (UL3-4), rHVT/IBD (UL22-23), and rHVT/IBD (UL45-46) exhibited decreased growth activity in chicken embryo fibroblast (CEF) cells compared to the parent HVT. However, the rHVT/IBD (US10) elicited lower levels of virus-neutralizing (VN) antibodies compared to the other constructs. rHVT/IBD (UL3-4) and rHVT/IBD (UL45-46) appeared to be similar in their ability to elicit VN antibodies. Based on the results of in vitro and in vivo assays, rHVT/IBD (UL3-4) was selected for further testing. In a challenge assay, rHVT/IBD (UL3-4) protected chickens from challenge with virulent Marek's disease virus serotype 1 and IBDV. In conclusion, the site of gene insertion may have a strong effect on the growth of the vector virus in vitro and its antibody-eliciting capacity. Insertions in the UL3-4 region permitted a balance between growth activity and VN-antibody-eliciting capacity, and this region might therefore be an appropriate insertion site for IBDV VP2.

Characterization of variant infectious bursal disease virus from a broiler farm in Japan using immunized sentinel chickens.

We attempted the isolation of variant infectious bursal disease (IBD) viruses by using sentinel chickens immunized with inactivated classical-type IBD vaccine. Immunized sentinel chickens with high levels of neutralizing antibodies and non-immunized sentinel chickens were raised together with broiler chickens in a commercial farm. Severe atrophy of the bursa of Fabricius was observed from the second week after cohabitation in non-immunized sentinel chickens. However, in immunized sentinel chickens and broiler chickens, atrophy was observed from the third week after cohabitation. The IBD virus (IBDV) isolated from the bursa of Fabricius of immunized sentinel chickens, designated as strain IBDV TY2, showed severe atrophy of the bursa in infected SPF chickens. Antiserum to the IBDV TY2 strain showed higher neutralizing activity to heterologous IBDV strains than did antiserum to the K strain vaccine virus. Phylogenetic analysis revealed that the nucleotide sequences encoding the hypervariable region of virus protein 2 of the IBDV TY2 strain did not cluster with the classical, variant or very virulent IBDV groups. Based on these results, we suggest that the IBDV TY2 strain may constitute a novel variant type of IBDV.

BioBrick-based 'Quick Gene Assembly' in vitro.

Because of the technological limitations of de novo DNA synthesis in (i) making constructs containing tandemly repeated DNA sequence units, (ii) making an unbiased DNA library containing DNA fragments with sequence multiplicity in a specific region of target genes, and (iii) replacing DNA fragments, development of efficient and reliable biochemical gene assembly methods is still anticipated. We succeeded in developing a biological standardized genetic parts that are flanked between a common upstream and downstream nucleotide sequences in an appropriate plasmid DNA vector (BioBrick)-based novel assembly method that can be used to assemble genes composed of 25 tandemly repeated BioBricks in the correct format in vitro. We named our new DNA part assembly system: 'Quick Gene Assembly (QGA)'. The time required for finishing a sequential fusion of five BioBricks is less than 24 h. We believe that the QGA method could be one of the best methods for 'gene construction based on engineering principles' at the present time, and is also a method suitable for automation in the near future.

Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome.

Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple ß-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15-80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10µg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose-response analysis indicated that a single immunization with as little as 1µg of the knob moiety of the CMP-knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens.

Isolation of endosulfan sulfate-degrading Rhodococcus koreensis strain S1-1 from endosulfan contaminated soil and identification of a novel metabolite, endosulfan diol monosulfate.

An aerobic endosulfan sulfate-degrading bacterium, Rhodococcus koreensis strain S1-1, was isolated from soil to which endosulfan had been applied annually for more than 10 years until 2008. The strain isolated in this work reduced the concentration of endosulfan sulfate (2) from 12.25 µM to 2.11 µM during 14 d at 30 °C. Using ultra performance liquid chromatography-electrospray ionization-mass spectroscopy (UPLC-ESI-MS), a new highly water-soluble metabolite possessing six chlorine atoms was found to be endosulfan diol monosulfate (6), derived from 2 by hydrolysis of the cyclic sulfate ester ring. The structure of 6 was elucidated by chemical synthesis of the candidate derivatives and by HR-MS and UPLC-MS analyses. Therefore, it was suggested that the strain S1-1 has a new metabolic pathway of 2. In addition, 6 was expected to be less toxic among the metabolites of 1 because of its higher water-solubility.

Decreased neutralizing antigenicity in IBV S1 protein expressed from mammalian cells.

We evaluated the antigenicity of recombinant infectious bronchitis virus (IBV) S1 protein expressed in mammalian cells. Recombinant S1 was expressed as a secreted protein fused with a trimerization motif peptide, then purified using Ni Sepharose. The purified protein was analyzed by Western blotting, mixed with oil adjuvant, and administered to 29-day-old specific-pathogen-free chickens. Six weeks after immunization, anti-IBV neutralizing titer and anti-S1 ELISA titer were determined; immunized chickens then were inoculated with IBV via the trachea and ciliary activity was observed. Results showed that the recombinant S1 protein was highly glycosylated, and the neutralizing antigenicity of recombinant S1 protein was lower than that of inactivated virus. However, anti-S1 ELISA indicated that the recombinant S1 protein induced antibodies against S1. These results suggest that the recombinant S1 may retain non-neutralizing epitopes but have unnatural glycosylation pattern and conformation, resulting in lacking neutralizing conformational epitopes. In conclusion, the neutralizing antigenicity of recombinant S1 protein expressed from mammalian cells was decreased, and was not sufficient to induce neutralizing antibodies.

Mineralization of melamine and cyanuric acid as sole nitrogen source by newly isolated Arthrobacter spp. using a soil-charcoal perfusion method.

Melamine belongs to the s-triazine family, and industrially used as raw product in many ways all over the world. Melamine has been reported for human harmful effects and detected from some crops, soil and water. To remove melamine from the polluted environment, the efficient melamine-mineralizing microorganisms have been needed. We newly isolated three melamine-degrading bacteria from the same upland soil sample using soil-charcoal perfusion method. These bacteria were classified as Arthrobacter sp. MCO, Arthrobacter sp. CSP and Microbacterium sp. ZEL by 16S rRNA genes sequencing analysis. Both Arthrobacter species completely degraded melamine within 2 days, and consumed melamine as a sole nitrogen source. Both strains also grew in cyanuric acid as sole nitrogen source, and released small quantities of ammonium ions. These strains are the first identified bacteria that can mineralize both melamine and cyanuric acid as sole initial nitrogen source in Arthrobacter sp. Although ammeline and ammelide intermediates were detected, these strains possess none of the known genes encoding melamine degrading enzymes. Since the Arthrobacter strains also degraded melamine in a high pH liquid medium, they present as potential bioremediation agents in melamine-polluted environments.

The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi.

Complex plant defenses that include the hypersensitive response (HR) are mediated by plant hormones, such as salicylic acid (SA), jasmonic acid (JA) and ethylene. We previously isolated the Arabidopsis DEAR1 (DREB AND EAR MOTIF PROTEIN 1) regulator and showed that its overexpression DEAR1 (DEAR1ox) resulted in a dwarf phenotype and lesion-like cell death, accompanied by elevated expression of PR (PATHOGENESIS-RELATED) genes. Here, we show that transgenic Arabidopsis overexpressing DEAR1 (DEAR1ox) has enhanced resistance to the necrotrophic fungus Botrytis cinerea (B. cinerea). This result indicates that DEAR1 represses negative regulators of plant defense responses, including transcriptional repressors belonging to the ERF (ETHYLEN RESPONSE FACTOR) family. Knockout mutants of ERF9 (erf9), which were down-regulated in DEAR1ox plants, showed transcriptional promotion of PDF1.2 (PATHOGEN-INDUCIBLE PLANT DEFENSIN) genes, which serve as positive markers for the ethylene/jasmonic acid (JA) signaling pathway and provide enhanced resistance to B. cinerea. Biochemical assays demonstrated that the ERF9 in capable of binding to the GCC box, a cis-element contained in the promoters of the PDF1.2 gene that possesses trans-repression activity. Moreover, infection with B. cinerea resulted in the promotion of the PDF1.2 expression, coinciding with suppression of the ERF9 gene under the control of the DEAR1 gene. These results indicate that the transcriptional repressor ERF9 participates in plant defense mechanisms against necrotic fungi mediated by the DEAR1-dependent ethylene/JA signaling pathway.

Relationship between severity of footpad dermatitis and carcass performance in broiler chickens.

The relationship between the severity of footpad dermatitis (FPD) and growth performance parameters (live weight, condemnation rate, leg meat yield and breast meat yield) was investigated in a total of 63 million broiler chickens that were processed over a period of 1,053 days between 2008 and 2012 at a full-scale processing plant in Kagoshima Prefecture, Japan. FPD scores and carcass data were summarized daily and analyzed to determine their correlations. It was found that FPD severity was positively correlated with the condemnation rate and negatively correlated with the live weight and leg meat yield. These results indicate that controlling FPD may play an important role in reducing condemnations while improving live weight and leg meat yields.

Determination of cellular aminopropyltransferase activity using precolumn fluorescent etheno-derivatization with high-performance liquid chromatography.

Polyamines such as spermidine (Spd) and spermine (Spm), produced by aminopropyltransferase (Apt), play roles in cell growth and differentiation. A sensitive and simple fluorometric high-performance liquid chromatographic determination for Apt activity of spermidine synthase (Spdsyn) and spermine synthase (Spmsyn) was developed in order to examine cellular functions of polyamine synthesis. The derivatization procedure for methylthioadenosine (MTA) produced from decarboxylated S-adenosylmethionine by Apt was the reaction with 2-chloroacetaldehyde to give fluorescent 1, N(6)-etheno methylthioadenosine. The reaction conditions for derivatization were optimized. A calibration curve was established, ranging from 0.01 to 25 pmol. Quantification of derivatized MTA was confirmed to be identical to Spd or Spm production. The developed method determined Spdsyn and Spmsyn activities in HepG2 cells treated with oleic acid as a cellular lipid accumulation model.

MBF1s regulate ABA-dependent germination of Arabidopsis seeds.

Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) controls gene expression by connecting transcription factors and the basal transcription machinery. In Arabidopsis thaliana functions of MBF1 genes have been related to stress tolerance and developmental alterations. Endogenous ABA plays a major role in the regulation of Arabidopsis seed dormancy and germination. Seed dormancy and ABA sensitivity are enhanced in ethylene insensitive mutants suggesting that ethylene signal transduction pathway is necessary to fully develop ABA-dependent germination. In this report we showed that a triple knock-down mutant for Arabidopsis MBF1 genes (abc-) has enhanced seed dormancy and displays hypersensitivity to exogenous ABA. In addition, higher ABA contents were detected in abc- seeds after imbibition. These evidences suggest a negative role of MBF1s genes in ABA-dependent inhibition of germination. The participation of MBF1s in ethylene signal transduction pathway is also discussed.

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species.

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil-charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.

Photodegradation of imidacloprid and fipronil in rice-paddy water.

Photodegradation of insecticides, imidacloprid and fipronil, in rice-paddy water under the ambient temperature was investigated. The initial concentrations were set at 58.8 and 3.1 µg/L for imidacloprid and fipronil, respectively, according to their reported initial concentrations in the rice-paddy field. The half-lives (DT(50)) of imidacloprid and fipronil were 24.2 and 36.7 h, respectively. Fipronil desulfinyl was detected as a major metabolite and fipronil sulfone was found to be a minor metabolite of fipronil in the photodegradation process. Detected mass of fipronil, fipronil desulfinyl, and fipronil sulfone at 79 h were 12.9%, 45.8%, and 5.2% of initial fipronil mass, respectively.

Footpad dermatitis in broiler chickens in Japan.

The prevalence of footpad dermatitis (FPD) in broiler chickens in Japan was investigated. In the first examination at slaughterhouses, lesions were commonly observed on the footpads of a total of 8,985 broiler chickens from 45 flocks on 36 farms. In 3 flocks, all the birds examined had lesions. In the other 42 flocks, the incidence of FPD ranged from 31.9% (81/254) to 99.5% (1/222). The footpad lesions were classified into 4 categories according to the severity of dermatitis as follows; score 0, 1,181 birds (13.1%); score 1, 2,992 birds (33.3%); score 2, 3,000 birds (33.4%); and score 3, 1,812 birds (20.2%). The mean scores of the flocks varied widely from 0.31 to 2.69. Males had higher mean scores than females. No significant differences were found between the mean scores of birds reared in windowless houses and those in open-sided houses. On 4 farms, the investigation was carried out in different seasons, and 3 of them showed higher mean scores in winter than in summer. Next, observation of 2,255 birds from 15 flocks on 5 farms during a 4-week rearing period revealed that FPD was already produced at 7 days of age and worsened with age.

The analysis of an Arabidopsis triple knock-down mutant reveals functions for MBF1 genes under oxidative stress conditions.

Transcriptional co-activators of the multiprotein bridging factor 1 (MBF1) type belong to a small multigenic family that controls gene expression by connecting transcription factors and the basal transcription machinery. In this report, a triple knock-down mutant (abc-) for the Arabidopsis thaliana MBF1 genes AtMBF1a, AtMBF1b and AtMBF1c was generated. The phenotypic characterization using oxidative agents such as hydrogen peroxide and methyl viologen revealed that the abc- mutant was more sensitive to oxidative stress. The triple knock-down mutant, abc- was also sensitive to osmotic stress mediated by high concentrations of sorbitol. Furthermore, the abc- phenotype was partially or completely rescued by AtMBF1c cDNA over-expression (abc- +c) depending on physiological and developmental conditions. AtMBF1s regulate the expression of ABR1, which is a member of the ethylene-response factor family and acts as ABA repressor. Thus, we conclude that AtMBF1 gene family may function as a regulatory component of the cross-talk node between ethylene, ABA and stress signal pathways. Furthermore, higher levels of a HSP70 mRNA and an immunoreactive HSP70 protein were detected in the abc- mutant. The participation of MBF1c as a possible negative regulator of HSP genes was discussed.

Intercomparison of induced fields in Japanese male model for ELF magnetic field exposures: effect of different computational methods and codes.

The present study provides an intercomparison of the induced quantities in a human model for uniform magnetic field exposures at extremely low frequency. A total of six research groups have cooperated in this joint intercomparison study. The computational conditions and numeric human phantom including the conductivity of tissue were set identically to focus on the uncertainty in computed fields. Differences in the maximal and 99th percentile value of the in situ electric field were less than 30 and 10 % except for the results of one group. Differences in the current density averaged over 1 cm(2) of the central nerve tissue are 10 % or less except for the results of one group. This comparison suggests that the computational uncertainty of the in situ electric field/current density due to different methods and coding is smaller than that caused by different human phantoms and the conductivitys of tissue, which was reported in a previous study.

Immune deviation and alleviation of allergic reactions in mice subjected to dietary restriction.

We examined cytokine production and allergic reactions in mice fed ad libitum (AL) and subjected to dietary restriction (DR). DR retarded the increase in body weight, and peripheral blood T cells in the DR mice produced less IFN-gamma and more IL-4 in response to immobilized anti-CD3 mAb. Systemic immunization and intranasal challenge with ovalbumin (OVA) induced accumulation of leukocytes into the lung, increase in IL-4 level in bronchoalveolar lavage fluid (BALF), and rise in serum IgE in the AL mice. In contrast, these allergic symptoms were alleviated in the DR mice. Furthermore, the relative proportion of IL-4-producing T cells responsive to OVA was less in the DR mice than the AL mice. DR tended to decrease the proportion and cytolytic activity of NK cells in the spleen, especially in younger mice. These results indicate that DR can prevent the expansion of allergen-specific IL-4-producing T cells followed by suppression of the allergic reaction, but might dampen NK cell activity.

Application of aluminum hydroxide for an in ovo live Newcastle disease vaccine.

Newcastle disease (ND) is a major threat to poultry, but the outbreak of the disease is well controlled by the vaccination. Recently, in ovo administration technology has been realized as a safe, efficacious, and convenient method for chicken vaccination. However, no in ovo administration has been applied for ND or other live vaccines that are highly pathogenic against chicken embryos. We found that an attenuated Newcastle disease virus (NDV) was applicable for an in ovo vaccination by adsorbing the virus to aluminum hydroxide (AH). Pathogenicity to chicken embryos of the AH-adsorbed NDV could be decreased compared with the administration of the virus alone. Namely, in ovo administration of the AH-adsorbed attenuated NDV resulted in improved hatchability and survival rate and better antibody responses of protection-level immunity compared with the administration of NDV alone. However, further improvements in hatchability and survival rate are necessary for practical application. From these results, in ovo vaccination with the AH-adsorbed attenuated NDV was revealed to be safe and immunogenic to chicken embryos. The use of AH-adsorbed attenuated live viruses might be applicable for in ovo vaccinations against not only ND but also other avian infectious diseases.

Arabidopsis MBF1s control leaf cell cycle and its expansion.

Multiprotein bridging factor 1 (MBF1) is known as a transcriptional co-activator that enhances transcription of its target genes by bridging between transcription factors and TATA-box-binding protein in eukaryotes. Arabidopsis thaliana has three MBF1 genes: AtMBF1a-AtMBF1c. However, details of the functions of AtMBF1 remain unclear. For this study, transgenic Arabidopsis overexpressing AtMBF1 fused to an active transcriptional repression domain (SRDX) was constructed. The chimeric protein putatively functions as a transcriptional co-repressor and as a suppressor of functions of endogenous AtMBF1 in transgenic plants. Transgenic Arabidopsis overexpressing AtMBF1-SRDX (AtMBF1-SRDX(OE)) showed an extremely small leaf phenotype under a continuous white light condition. Its leaf cells-especially those around vascular tissues, where strong expression of endogenous AtMBF1s is observed-were much smaller than those from the wild type (WT). In addition, a lower cell number was observed in leaves from AtMBF1-SRDX(OE) plants. Time course analysis of cell size revealed that cell expansion of leaves of AtMBF1-SRDX(OE) plants was dramatically suppressed during the late leaf developmental stage (cell expansion stage), when endogenous AtMBF1b is strongly expressed in the WT. The results show that ploidy levels of leaves from AtMBF1-SRDX(OE) plants were dramatically lower than those from the WT; moreover, expression levels of several negative regulators of endoreduplication were more elevated in AtMBF1s-SRDX(OE) plants than those in the WT. These observations suggest that AtMBF1-SRDX interacts with regulators of endoreduplication. Therefore, AtMBF1s are considered to affect not only leaf cell expansion but also regulation of the ploidy level in leaf cells during the leaf expansion stage.