Safety Concerns with Nusinersen, Risdiplam, and Onasemnogene Abeparvovec in Spinal Muscular Atrophy: A Real-World Pharmacovigilance Study.

BACKGROUND AND OBJECTIVE: Spinal muscular atrophy (SMA) is a genetic disorder with limited treatment options. It is crucial to have a comprehensive understanding of drug safety in order to make informed clinical drug selections for patients with SMA. Assessing the safety profiles of therapeutic drugs for SMA has been challenging due to the limited number of patients included in clinical trials. This study aims to investigate and compare the potential safety concerns associated with three leading SMA therapeutic drugs: nusinersen, risdiplam, and onasemnogene abeparvovec. METHODS: The FDA Adverse Event Reporting System database was used to analyze drug safety, and a case (SMA drug)/noncase (all other drugs in the database) approach was employed to estimate safety signals through disproportionality analysis and reporting odds ratio (ROR). Veen analysis was conducted to compare and select the idiosyncratic adverse events (AEs) associated with each drug. RESULTS: The study included 5324 cases of nusinersen, 1184 cases of risdiplam, and 1277 cases of onasemnogene abeparvovec. Venn analysis revealed 27 common AEs among the three drugs, including cardiac, gastrointestinal, metabolism, musculoskeletal, renal, respiratory disorders, and infections. Additionally, 196 AEs exclusively found in nusinersen included post lumbar puncture syndrome [ROR (95% CI) = 6120.91 (5057.01-7408.64), n = 372], procedural pain [ROR (95% CI) = 54.86 (48.13-62.54), n = 234], idiopathic intracranial hypertension [ROR (95% CI) = 6.12 (2.29-16.33), n = 4], and hypokalemia [ROR (95% CI) = 2.02 (1.24-3.31), n = 16]. Additionally, transient deafness was identified as an unexpected and rare, yet severe, AE for nusinersen [ROR (95% CI) = 23.32 (8.71-62.44), n = 4]. Risdiplam exhibited 50 AEs exclusively, with notable idiosyncratic AEs including diarrhea [ROR (95% CI) = 4.55 (3.79-5.46), n = 121], fatigue [ROR (95% CI) = 2.03 (1.6-2.57), n = 70], photosensitivity reaction [ROR (95% CI) = 9.50 (4.25-21.13), n = 6], rash [ROR (95% CI) = 1.90 (1.36-2.67), n = 34], and [ROR (95% CI) = 4.3 (1.93-9.58), n = 6] in comparison with the other two drugs. Moreover, ileus [ROR (95% CI) = 11.11 (4.14-29.51), n = 4], gastrointestinal hemorrhage [ROR (95% CI) = 2.55 (1.15-5.69), n = 6], and hypoglycemic unconsciousness [ROR (95% CI) = 153.58 (62.98-374.54), n = 5] were rare but severe AEs associated with risdiplam. Onasemnogene abeparvovec had 143 exclusively identified AEs, with significant high signals for troponin I increase [ROR (95% CI) = 627.1 (492.2-798.99), n = 78], troponin T increase [ROR (95% CI) = 233.98 (153.29-357.15), n = 23], blood lactate dehydrogenase increase [ROR (95% CI) = 39.81 (28.88-54.87), n = 38], and transaminases increase [ROR (95% CI) = 36.88 (29.24-46.52), n = 73]. CONCLUSIONS: This study highlights the importance of monitoring injection-related injuries and transient deafness events in patients treated with nusinersen. For onasemnogene abeparvovec, careful monitoring for renal impairment, liver injury, and myocardial damage is necessary. Risdiplam requires attention to the potential risk of rare but severe gastrointestinal damage events and hypoglycemia. Importantly, risdiplam exhibited lower liver and renal toxicity, making it a potential consideration for patients with liver or renal insufficiency or for combined use with other drugs that possess high liver or renal toxicity. These findings can be a reference for drug selection and further prospective studies.

Preparation of Polyacrylonitrile-Based Immobilized Copper-Ion Affinity Membranes for Protein Adsorption.

A polyacrylonitrile (PAN)-based immobilized metal-ion affinity membrane (IMAM) was prepared with a high capacity for protein adsorption. PAN was selected as the substrate due to its excellent thermal and chemical stability. The cyano groups on the PAN membrane were substituted with carboxyl groups, followed by reactions with ethylenediamine (EDA) and ethylene glycol diglycidyl ether (EGDGE) to produce the terminal epoxy groups. The chelating agent iminodiacetic acid (IDA) was then bound to the modified PAN membrane and further chelated with copper ions. The immobilized copper ion amount of membrane was analyzed to obtain the optimal reaction conditions, which were 60 °C/3 h for EDA coupling and 60 °C/4 h for EGDGE grafting. Furthermore, under the use of minor IDA and copper ion concentrations, the immobilized copper ion capacity of the IMAM was 4.8 µmol/cm2 (253.4 µmol/mL, or 1.47 µmol/mg). At a neutral pH, the cationic lysozyme exhibited a large adsorption capacity with the IMAM (1.96 µmol/mL), which was most likely multilayer binding, whereas the adsorption capacity for bovine serum albumin (BSA) and histidine-tagged green fluorescent protein (GFP-His6) was 0.053 µmol/mL and 0.135 µmol/mL, respectively, with a monolayer adsorption arrangement. The protein desorption efficiency was greater than 95%, implying that the prepared IMAM could be reused for protein adsorption.

Triptolide attenuates LPS-induced activation of RAW 264.7 macrophages by inducing M1-to-M2 repolarization via the mTOR/STAT3 signaling.

CONTEXT: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of gastrointestinal tract, which can develop into colorectal cancer. Triptolide (TP) is a predominant bioactive ingredient of Tripterygium wilfordii Hook.F., and has been proven to have the therapeutic potential for various human diseases. OBJECTIVE: In our study, we examined the function of TP in the progression of IBD. METHODS: 3-(4,5)dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide assay was used to evaluate the viability of RAW264.7 cells. Quantitative reverse transcription polymerase chain reaction assay was performed to detect the relative gene expression. Western blot was used to detect the relative protein expression. Enzyme-linked immunosorbent assay was utilized to examine the levels of prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-10, and IL-6. RESULT: Our research demonstrated that TP restrained lipopolysaccharide (LPS)-caused activation of RAW264.7 cells, as evidenced by the reduction of PGE2, TNF-α, and IL-6, and increase of IL-10. TP treatment also restrained M1-type macrophage polarization and facilitated M2-type macrophage polarization of RAW 264.7 cells in the presence of LPS. Moreover, TP mitigated LPS-induced activation of the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling in RAW264.7 cells. Further, activation of the mTOR/STAT3 signaling by MHY1485 attenuated the effect of TP in regulation of macrophage polarization in RAW264.7 cells in the presence of LPS. CONCLUSION: Overall, our results indicated that TP attenuated LPS-induced activation of RAW 264.7 macrophages by inducing M1-to-M2 repolarization via repression of the mTOR/STAT3 signaling. Therefore, TP might be an effective agent for IBD treatment.

Tanshinol alleviates ulcerative colitis by promoting the expression of VLDLR.

Tanshinol (TAN) is a widely used Chinese medicine ingredient with anti-inflammatory activity. The therapeutic effect of TAN in ulcerative colitis (UC) deserves further investigation. DSS induced UC model for mice, and TAN of different concentrations was used for in vivo therapy. Colons length was measured; expression of VLDLR in colonic mucosal tissue was evaluated by qRT-PCR, Western blot and histochemical staining. Besides, normal colorectal mucosal cell line (FHC) was treated with LPS to imitate the inflammatory process of UC in vitro. Different concentrations of TAN treated UC cell model. ELISA and qRT-PCR were applied to examine the concentrations of inflammatory cytokines (TNF-α, IL-6, IL-8, or IL-1ß). Flow cytometry and MTT was used to identify the apoptosis and viability of FHC cells, respectively. Afterwards, Western blot was performed to detect the expressions of Bax, Bcl-2, Cleaved caspase-3, and Cleaved caspase-9 in FHC cells. VLDLR was low-expressed in UC tissues as compared to the normal tissue. TAN could alleviate DSS-induced colons length shortening, colonic tissue structure destruction, inflammatory response, and VLDLR expression decrease in vivo. Further study found that TAN could alleviate LPS-induced inflammatory response, apoptosis, and viability decrease of FHC cells, and siVLDLR could partially offset the effect of TAN. TAN alleviates LPS-induced viability decrease, apoptosis, and inflammatory response in FHC cells by promoting VLDLR expression.

Phagocytosis activity of three sulfated polysaccharides purified from a marine diatom cultured in a semi-continuous system.

An efficient process has been developed for bioactive polysaccharide production and purification from a local diatom isolate, Halamphora sp. AQ4. First, a semi-continuous system with fixed harvesting frequency was employed to cultivate AQ4 for the production of cell mass and polysaccharides for more than 285 days with a high yield of biomass. Six cultivation sets are performed according to different harvesting volumes per 3 days with or without Na2CO3 supplement. The addition of Na2CO3 increases both cell mass and polysaccharide production. Furthermore, three different sulfated polysaccharides (PK1~PK3) were purified from the freshly-grown AQ4 diatoms following anion-exchange chromatography. Among them, polysaccharide PK3 not only has a high content of fucose and uronic acid, but also has a strong activity to stimulate murine macrophage cells and increase their phagocytosis rate up to 170%. This study demonstrates that diatom AQ4 is an important bioresource for the production of bioactive polysaccharides.

Purification of lysozyme from chicken egg white using diatom frustules.

A nontoxic chromatographic matrix, with low cost and high adsorption capacity, is always a major goal for therapeutic protein purification. In this study, the frustules from two cultured diatoms, Nitzschia bilobata (AQ1) and Psammodictyon panduriforme (NP), were investigated as cation exchange materials for lysozyme purification from chicken egg white. The surface area and cation exchange capacity of frustules were about 400 m2/g and 140 µmol/mL for AQ1, 390 m2/g and 130 µmol/mL for NP. The optimal pH was 9 for adsorption. Through batch purification, the lysozyme recovery was 86% with a purity of 95% by AQ1 frustules, which was higher than that by NP frustules (82% with a purity of 90%). In the flow-through system, the purity using AQ1 frustules notably increased to 99%, higher than the result of 91% using NP frustules. Diatom frustules from AQ1 are more effective and could be an alternative chromatographic matrix for lysozyme purification.

[Effect of glucocorticoid receptor function on the behavior of rats with attention deficit hyperactivity disorder].

OBJECTIVE: To investigate the ideal animal models for attention deficit hyperactivity disorder (ADHD) subtypes and the effect of glucocorticoid receptor (GR) function on the behavior of ADHD rats by comparing behavioral differences between spontaneously hypertensive rats (SHRs), Wistar Kyoto (WKY) rats, and Sprague-Dawley (SD) rats. METHODS: A total of 24 male SHRs aged 21 days were randomly divided into GR agonist group, GR inhibitor group, and SHR group, with 8 rats in each group. Eight male WKY rats and 8 male SD rats, also aged 21 days, were enrolled as WKY group and SD group respectively. The GR agonist group was treated with intraperitoneal injection of dexamethasone (0.5 mg/kg daily); the GR inhibitor group was treated with intraperitoneal injection of mifepristone (RU486) (54 mg/kg daily); the SHR, WKY, and SD groups were treated with intraperitoneal injection of normal saline (0.5 mL/kg daily). The course of treatment was 14 days for all groups. The open field test and Lat maze test were used to evaluate spontaneous activity and non-selective attention. RESULTS: The open field test showed that before drug intervention the SHR group had significantly higher numbers of line crossings and rearings than the WKY and SD groups (P<0.05); the WKY group had a significantly higher number of line crossings than the SD group (P<0.05); the SD group had a significantly higher number of groomings than the WKY group (P<0.05). After drug intervention, the GR agonist group had significantly lower numbers of line crossings and groomings than the SHR group (P<0.05). The Lat maze test indicated that before drug intervention the SHR group had significantly higher numbers of corner crossings and rearings than the WKY and SD groups (P<0.05); the WKY group had significantly higher numbers of rearings and leanings than the SD group (P<0.05). After drug intervention, the GR agonist group had significantly lower numbers of corner crossings and rearings than the SHR group (P<0.05); the GR inhibitor group had a significantly higher number of rearings than the SHR group (P<0.05); the WKY group had significantly higher numbers of rearings and leanings than the SD group (P<0.05). CONCLUSIONS: SHR is an ideal animal model for mixed subtype ADHD, and further studies are needed to determine whether WKY rats can be used as an animal model for attention-deficit subtype ADHD. GR agonist can effectively improve spontaneous activity and non-selective attention in SHRs.

Valence processing of first impressions in the dorsomedial prefrontal cortex: a near-infrared spectroscopy study.

Previous studies have suggested that the dorsomedial prefrontal cortex (dmPFC) plays a central role in processing first impressions; however, little is known about how dmPFC processes different valences of first impressions. Moreover, it is still unclear as to whether the dmPFC shows lateralization or only induces different levels of activation when processing positive and negative impressions. To address these questions in the present study, the brain activities for the impression judgments expressed by participants were measured with near-infrared spectroscopy. For each real facial picture, participants were asked to evaluate their first impressions on a scale from 'bad' to 'good' using a keyboard. The results showed that although the right dmPFC has a higher sensitivity in processing impressions, both the hemispheres of dmPFC showed a significant trend where the activation of positive impressions was higher than the negative ones. Accordingly, it is proposed that the dmPFC acts as a single mechanism responsible for delineating the processing of first impressions rather than two lateralized systems. Therefore, a 'positivity dominance hypothesis' is also proposed, which states that dmPFC in both hemispheres have a higher sensitivity and priority for positive impressions than negative ones. The present study provides valuable findings with respect to the role of the dmPFC in the processes of first impression formation.

p53 is a key regulator for osthole-triggered cancer pathogenesis.

Osthole has been reported to have antitumor activities via the induction of apoptosis and inhibition of cancer cell growth and metastasis. However, the detailed molecular mechanisms underlying the anticancer effects of osthole in human colon cancer remain unclear. In the present study, we have assessed osthole-induced cell death in two different human colon cancer cell lines, HCT116 and SW480. Our results also showed that osthole activated proapoptotic signaling pathways in human colon cancer cells. By using cell culture insert system, osthole reduced cell motility in both human colon cancer cell lines. This study also provides evidence supporting the potential of osthole in p53 activation. Expression of p53, an apoptotic protein, was remarkably upregulated in cells treated with osthole. Importantly, the levels of phosphorylation of p53 on Ser15 (p-p53) and acetylation of p53 on Lys379 (acetyl-p53) were increased under osthole treatment. Our results also demonstrated that p53 was activated followed by generation of reactive oxygen species (ROS) and activation of c-Jun N-terminal kinase (JNK). Our study provides novel insights of p53-mediated responses under osthole treatment. Taken together, we concluded that osthole induces cancer cell death and inhibits migratory activity in a controlled manner and is a promising candidate for antitumor drug development.

A single amino acid in VP2 is critical for the attachment of infectious bursal disease subviral particles to immobilized metal ions and DF-1 cells.

VP2 protein is the primary host-protective immunogen of infectious bursal disease virus (IBDV). His249 and His253 are two surface histidine residues in IBDV subviral particles (SVP), which is formed by twenty VP2 trimers when the VP2 protein of a local isolate is expressed. Here, a systemic study was performed to investigate His249 or/and His253 on self-assembly, cell attachment and immunogenicity of SVP. Point-mutagenesis of either or both histidine residues to alanine did not affect self-assembly of the SVP, but the SVP lost its Ni-NTA binding affinity when the His253 was mutated. Indirect immunofluorescence assays and inhibitory experiments also showed that His253 is essential for SVP to attach onto the DF-1 cells and to inhibit IBDV infection of DF-1 cells. Finally, enzyme-linked immunosorbent assays and chicken protection assays demonstrated that SVP with a mutation of His253 to alanine induced comparable neutralizing antibody titers in chickens as the wild-type SVP did. It was concluded that VP2's His253, a site not significant for the overall immunogenicity induced by SVP, is crucial for the binding affinity of SVP to Ni-NTA and the attachment of an IBDV host cell line. This is the first paper to decipher the role of His253 played in receptor interaction and immunogenicity.

GDNF increases cell motility in human colon cancer through VEGF-VEGFR1 interaction.

Glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor, has been shown to affect cancer cell metastasis and invasion. However, the molecular mechanisms underlying GDNF-induced colon cancer cell migration remain unclear. GDNF is found to be positively correlated with malignancy in human colon cancer patients. The migratory activities of two human colon cancer cell lines, HCT116 and SW480, were found to be enhanced in the presence of human GDNF. The expression of vascular endothelial growth factor (VEGF) was also increased in response to GDNF stimulation, along with VEGF mRNA expression and transcriptional activity. The enhancement of GDNF-induced cancer cell migration was antagonized by a VEGF-neutralizing antibody. Our results also showed that the expression of VEGF receptor 1 (VEGFR1) was increased in response to GDNF stimulation, whereas GDNF-induced cancer cell migration was reduced by a VEGFR inhibitor. The GDNF-induced VEGF expression was regulated by the p38 and PI3K/Akt signaling pathways. Treatment with GDNF increased nuclear hypoxia-inducible factor 1 α (HIF1α) accumulation and its transcriptional activity in a time-dependent manner. Moreover, GDNF increased hypoxia responsive element (HRE)-containing VEGF promoter transcriptional activity but not that of the HRE-deletion VEGF promoter construct. Inhibition of HIF1α by a pharmacological inhibitor or dominant-negative mutant reduced the GDNF-induced migratory activity in human colon cancer cells. These results indicate that GDNF enhances the migration of colon cancer cells by increasing VEGF-VEGFR interaction, which is mainly regulated by the p38, PI3K/Akt, and HIF1α signaling pathways.

Endopeptidase activity characterization of E. coli-derived infectious bursal disease virus protein 4 tubules.

Viral protein 4 (VP4) is a serine protease that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 of infectious bursal disease virus. In this report, the recombinant VP4 with a His-tag and three mutants (VP4-S652A, VP4-K692A and VP4-S652A.K692A) were expressed in Escherichia coli. Soluble VP4 was purified using immobilized metal-ion affinity chromatography or sucrose density gradient following with gel-filtration chromatography. The purified VP4 has a tubular structure with 25-30 nm in width and ∼300 nm in length, as observed by transmission electron microscope. A similar tubular structure was also found for these three mutants. The endopeptidase activity of these VP4 tubules was characterized by fluorescence resonance energy transfer using a synthetic fluorogenic oligopeptide as a substrate. The results show that the tubule-like VP4 is a functional enzyme with K(m) of 43 ± 2 µM and k(cat) of 0.04 ± 0.01 min⁻¹; however, k(cat) of three mutants were significantly reduced. This is the first report to demonstrate that VP4 protein expressed in E. coli can self-assemble into functional tubule-like particles and its activity can be completely inhibited by 1 mM of Ni⁺² ions.

Development of an anti-influenza drug screening assay targeting nucleoproteins with tryptophan fluorescence quenching.

Recent studies have shown that NP (nucleoprotein), which possesses multiple functions in the viral life cycle, is a new potential anti-influenza drug target. NP inhibitors reliably induce conformational changes in NPs, and these changes may confer inhibition of the influenza virus. The six conserved tryptophan residues in NP can be used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residues in the protein upon binding to an NP inhibitor. In the present study, we found that the fluorescence of recombinant NP proteins was quenched following the binding of available NP inhibitors (such as nucleozin) in a concentration- and time-dependent manner, which suggests that the inhibitor induced conformational changes in the NPs. The minimal fluorescence-quenching effect and weak binding constant of nucleozin to the swine-origin influenza virus H1N1pdm09 (SOIV) NP revealed that the SOIV is resistant to nucleozin. We have used the fluorescence-quenching property of tryptophans in NPs that were bound to ligands in a 96-well-plate-based drug screen to assess the ability of promising small molecules to interact with NPs and have identified one new anti-influenza drug, CSV0C001018, with a high SI value. This convenient method for drug screening may facilitate the development of antiviral drugs that target viruses other than the influenza virus, such as HIV and HBV.

Induction of protective immunity in chickens immunized with plant-made chimeric Bamboo mosaic virus particles expressing very virulent Infectious bursal disease virus antigen.

Very virulent Infectious bursal disease virus (vvIBDV) causes a highly contagious disease in young chickens and leads to significant economic loss in the poultry industry. Effective new vaccines are urgently needed. Autonomously replicating plant virus-based vector provides attractive means for producing chimeric virus particles (CVPs) in plants that can be developed into vaccines. In this study, we demonstrate the potential for vaccine development of Bamboo mosaic virus (BaMV) epitope-presentation system, where the antigen from vvIBDV VP2 was fused to the N-terminus of BaMV coat protein. Accordingly, an infections plasmid, pBIBD2, was constructed. Inoculation of the recombinant BaMV clone pBIBD2 enabled the generation of chimeric virus, BIBD2, and stable expression of IBDV VP2 antigen on its coat protein. After intramuscular immunization with BIBD2 CVPs, chickens produced antibodies against IBDV and were protected from vvIBDV (V263/TW strain) challenges. These results corroborate the feasibility of BaMV-based CVP platform in plants for the development and production of vaccines against IBDV.

One-step reverse-transcription loop-mediated isothermal amplification for detection of infectious bursal disease virus.

A fast, sensitive, and specific reverse-transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay was developed that involved a single tube and a 1-step reaction for detecting infectious bursal disease virus (IBDV). Four specific primers were used for amplification of the VP2 gene of IBDV. The amplified LAMP products were detected by DNA electrophoresis and by direct observation with the naked eye in the presence of SYBR Green I. The sensitivity of RT-LAMP was determined to be 0.01 fg of IBDV viral RNA. This assay for IBDV is more sensitive than the conventional RT-polymerase chain reaction assay, which has a detection limit of 1 ng. The LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. This newly established LAMP assay, combined with RT, is a practical diagnostic tool because IBDV-infected and uninfected clinical samples collected from an experimental farm could be discriminated. Full verification of a sample's IBDV status was obtained within 40 min of extraction of the viral RNA, which could then be directly added to the RT-LAMP reaction mixture.

High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development.

BACKGROUND: Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention. RESULTS: Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay. CONCLUSIONS: Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.

Development of a disperse dye immunochromatographic test for the detection of antibodies against infectious bursal disease virus.

For investigating the feasibility of using disperse dyes as an immunoassay chromogenic marker, a disperse dye, DADISPERSE NAVY BLUE SP, was selected in analyzing antibody against infectious bursal disease virus (anti-IBDV). With the color intensity revealed in the disperse dye immunochromatographic test (DICT) strip as the objective function, the optimal dyeing conditions were found as follows: dye concentration absorbance (at lambda(max)=587nm)=3, pH 7, 50 degrees C, for 10min. Under these conditions, the resultant dyed-antibody (rabbit anti-chicken) can produce an optimal color intensity reading of 55,054 on the strip. For performing qualitative immunoassay, chicken sera samples taken from different farms were used for the anti-IBDV titre assessment. The results of DICT strips showed very high sensitivity and specificity as compared to that analyzed by FlockChek enzyme linked immunosorbent assay (F-ELISA) kits. For quantitative immunoassay, it was found that the color intensity measured with DICT was linearly correlated to that of F-ELISA titre (r(2)=0.9687). Therefore, DICT was further applied to the detection of chicken anti-IBDV sera under vaccination in the farms. The average titres of the sampling groups exhibited a strong agreement to that of F-ELISA. Accordingly, the DICT method developed in this study, shown to be reliable, cheap and simple in both qualitative and quantitative immunoassays, is particularly suitable for point-of-need testing (PONT) in agricultural applications.

Development of an enzyme-linked immunosorbent assay for detecting infectious bursal disease virus (IBDV) infection based on the VP3 structural protein.

Infectious bursal disease virus (IBDV) causes a contagious immunosuppressive disease in chickens. The aim of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using the expressed VP2 or VP3 protein of IBDV as the coating antigen for detecting antibodies to IBDV. Experimental results were compared with virus neutralization assay and a commercial-available ELISA. These assays were used to examine the sera from farm chickens and chickens vaccinated experimentally. The VP3-based ELISA had a higher correlation coefficient (R2) of 0.812 with a commercial ELISA kit at a serum dilution of 1:500 than that of VP2-based ELISA (R2) of 0.671. The relative sensitivity between virus neutralization and VP2-ELISA and VP3-ELISA was 96% (251/262) and 100% (262/262), respectively, and that between virus neutralization and a commercial ELISA was 99% (257/261). Additionally, compared with virus neutralization assay, the reference technique for diagnosing IBDV, VP3-based ELISA had an agreement value of 99%, superior to that of VP2-based ELISA (95%) or the commercial kit (89%). These results revealed that the capability of either VP2-ELISA or VP3-ELISA in detecting the field chicken sera was comparable to the commercial one, which is generally used to replace the virus neutralization assay. However, the preparation of VP3 is derived from an Escherichia coli expression system with a high yield and purification efficiency by Ni2+-NTA gels, which is more favorable to the insect cell-derived particles formed by VP2. Therefore, VP3-ELISA could be developed as an efficient and low cost diagnostic method for IBDV infection in field chickens.

Strong and heterogeneous adsorption of infectious bursal disease VP2 subviral particle with immobilized metal ions dependent on two surface histidine residues.

VP2, the single outer protein of infectious bursal disease virus capsid, can self-assemble into T = 1 subviral particle (SVP), which can be efficiently purified by immobilized metal ion affinity chromatography (IMAC). In this study, a systemic investigation of the adsorption behavior of VP2 SVP on Ni-NTA resin was performed to identify that His253 and His249 on the surface of SVP are the key factors accounted for the strong and heterogeneous interaction. First, an untagged VP2-441 SVP was constructed, expressed, and purified by IMAC to demonstrate that SVP can interact with immobilized Ni2+ ions on NTA resin without an inserted His tag. Second, equilibrium adsorption studies were used to demonstrate that SVP has a higher affinity to the immobilized Ni2+ ions than a model protein, bovine serum albumin, although the maximum amount of SVP bound per volume resin is limited by the pore size of the resin as verified by confocal microscopic analysis. Third, based on structural analysis and computer modeling, His253 and His249 on the surface of SVP are responsible for a strong heterogeneous and multiple adsorption with the immobilized Ni2+ ions; and this was confirmed by a point-mutation experiment. This is the first example to elucidate the interaction between the immobilized metal ions and viral particles at molecular level. A detailed understanding of SVP-immobilized metal ion interactions can provide useful strategies for engineering icosahedral protein nanoparticles to achieve a simple and one-step purification by IMAC.

Chicken heat shock protein 90 is a component of the putative cellular receptor complex of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. The capsid protein VP2 of IBDV plays an important role in virus binding and cell recognition. VP2 forms a subviral particle (SVP) with immunogenicity similar to that of the IBDV capsid. In the present study, we first showed that SVP could inhibit IBDV infection to an IBDV-susceptible cell line, DF-1 cells, in a dose-dependent manner. Second, the localizations of the SVP on the surface of DF-1 cells were confirmed by fluorescence microscopy, and the specific binding of the SVP to DF-1 cells occurred in a dose-dependent manner. Furthermore, the attachment of SVP to DF-1 cells was inhibited by an SVP-induced neutralizing monoclonal antibody against IBDV but not by denatured-VP2-induced polyclonal antibodies. Third, the cellular factors in DF-1 cells involved in the attachment of SVP were purified by affinity chromatography using SVP bound on the immobilized Ni(2+) ions. A dominant factor was identified as being chicken heat shock protein 90 (Hsp90) (cHsp90) by mass spectrometry. Results of biotinylation experiments and indirect fluorescence assays indicated that cHsp90 is located on the surface of DF-1 cells. Virus overlay protein binding assays and far-Western assays also concluded that cHsp90 interacts with IBDV and SVP, respectively. Finally, both Hsp90 and anti-Hsp90 can inhibit the infection of DF-1 cells by IBDV. Taken together, for the first time, our results suggest that cHsp90 is part of the putative cellular receptor complex essential for IBDV entry into DF-1 cells.