Expression of VP2 protein of Novel goose parvovirus in baculovirus and evaluation of its immune effect.

Short-beak and dwarfism syndrome (SBDS) is a new disease caused by a genetic variant of goose parvovirus in ducks that results in enormous economic losses for the waterfowl industry. Currently, there is no commercial vaccine for this disease, so it is urgent to develop a safer and more effective vaccine to prevent this disease. In this study, we optimized the production conditions to enhance the expression of the recombinant VP2 protein and identified the optimal conditions for subsequent large-scale expression. Furthermore, the protein underwent purification via nickel column affinity chromatography, followed by concentration using ultrafiltration tube. Subsequently, it was observed by transmission electron microscopy (TEM) that the NGPV recombinant VP2 protein assembled into virus-like particles (VLPs) resembling those of the original virus. Finally, the ISA 78-VG adjuvant was mixed with the NGPV-VP2 VLPs to be prepared as a subunit vaccine. Furthermore, both agar gel precipitation test (AGP) and serum neutralization test demonstrated that NGPV VLP subunit vaccine could induce the increase of NGPV antibody in breeding ducks. The ducklings were also challenged with the NGPV, and the results showed that the maternal antibody level could provide sufficient protection to the ducklings. These results indicated that the use of the NGPV VLP subunit vaccine based on the baculovirus expression system could facilitate the large-scale development of a reliable vaccine in the future.

Transcriptomic analysis of ncRNAs and mRNAs interactions during drought stress in switchgrass.

Switchgrass (Panicum virgatum L.) plays a pivotal role as a bioenergy feedstock in the production of cellulosic ethanol and contributes significantly to enhancing ecological grasslands and soil quality. The utilization of non-coding RNAs (ncRNAs) has gained momentum in deciphering the intricate genetic responses to abiotic stress in various plant species. Nevertheless, the current research landscape lacks a comprehensive exploration of the responses of diverse ncRNAs, including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs), to drought stress in switchgrass. In this study, we employed whole transcriptome sequencing to comprehensively characterize the expression profiles of both mRNA and ncRNAs during episodes of drought stress in switchgrass. Our analysis identified a total of 12,511 mRNAs, 59 miRNAs, 38 circRNAs, and 368 lncRNAs that exhibited significant differential expression between normal and drought-treated switchgrass leaves. Notably, the majority of up-regulated mRNAs displayed pronounced enrichment within the starch and sucrose metabolism pathway, as validated through KEGG analysis. Co-expression analysis illuminated that differentially expressed (DE) lncRNAs conceivably regulated 1308 protein-coding genes in trans and 7110 protein-coding genes in cis. Furthermore, both cis- and trans-target mRNAs of DE lncRNAs exhibited enrichment in four common KEGG pathways. The intricate interplay between lncRNAs and circRNAs with miRNAs via miRNA response elements was explored within the competitive endogenous RNA (ceRNA) network framework. As a result, we constructed elaborate regulatory networks, including lncRNA-novel_miRNA480-mRNA, lncRNA-novel_miRNA304-mRNA, lncRNA/circRNA-novel_miRNA122-PvSS4, and lncRNA/circRNA-novel_miRNA14-PvSS4, and subsequently validated the functionality of the target gene, starch synthase 4 (PvSS4). Furthermore, through the overexpression of PvSS4, we ascertained its capacity to enhance drought tolerance in yeast. However, it is noteworthy that PvSS4 did not exhibit any discernible impact under salt stress conditions. These findings, as presented herein, not only contribute substantively to our understanding of ceRNA networks but also offer a basis for further investigations into their potential functions in response to drought stress in switchgrass.

Erastin inhibits porcine epidemic diarrhea virus replication in Vero cells.

Background: Porcine epidemic diarrhea virus (PEDV), an intestinal pathogenic coronavirus, has caused significant economic losses to the swine industry worldwide. At present, there are several treatment methods, but there is still a lack of clinically effective targeted drugs, new antiviral mechanisms and drugs need to be explored. Methods: In this study, we established a model of erastin versus ferrostatin-1 treatment of Vero cells, and then detected virus proliferation and gene expression by RT-qPCR through PEDV infection experiments. Results: We demonstrated for the first time that erastin significantly inhibited the replication of PEDV upon entry into cells; Vero treated with erastin significantly regulated the expression of three genes, NRF2, ACSL4 and GPX4, notably erastin regulated the expression of these three genes negatively correlated with the expression induced by PEDV virus infection. Conclusions: Since NRF2, ACSL4 and GPX4 are classical Ferroptosis genes, this study speculates that erastin may inhibit the replication of PEDV in Vero cells in part through the regulation of ferroptosis pathway, and erastin may be a potential drug for the treatment of PEDV infection.

A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies.

Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodies derived from camelids with naturally missing light chains can overcome these deficiencies and are an excellent alternative to conventional antibodies. In this study, a nanobody (Nb)-AviTag fusion protein was constructed, and the feasibility of its use as a high-sensitivity probe in a blocking ELISA (bELISA) for classical swine fever virus (CSFV) was investigated. The CSFV E2 recombinant protein expressed by the CHO expression system exhibited good reactogenicity and immunogenicity and induced the production of high CSFV antibody levels in rabbits. Three different clones of Nbs were successfully isolated using a phage display system in alpaca, and an Nb1-AviTag fusion protein was successfully expressed using an Escherichia coli expression system. The purified Nb1-AviTag fusion protein was then biotinylated in vitro to obtain Nb1-biotin. A novel bELISA was developed for the detection of CSFV antibodies in clinical serum using Nb1-biotin as a probe. The cutoff value of bELISA was 32.18%, the sensitivity of bELISA was higher than that of the bELISA kit with IDEXX antibody, and the coincidence rate was 94.7%. A rapid, low-cost, highly sensitive and highly specific CSFV E2 antibody-based bELISA method was successfully established and can be used for the serological evaluation of CSFV E2 subunit vaccines and the ELISA-based diagnosis of CSFV infection. IMPORTANCE Currently, the epidemic situation of classical swine fever (CSF) is sporadic, and cases of atypical swine fever are on the rise in China. Therefore, it is necessary to accurately eliminate suspected cases by using highly sensitive and specific diagnostic techniques. In our study, a rapid, low-cost, highly sensitivity, highly reliable and reproducible, and highly specific classical swine fever virus (CSFV) E2 antibody-based blocking ELISA method was successfully established by using the phage display system and the Nb1-AviTag fusion expression platform. It provides a new technique for serological evaluation of CSFV vaccines and ELISA-based diagnosis of CSFV infection.

Ratiometric detection of transcription factor based on Europium(III) complex-doped silicon nanoparticles and a G-quadruplex-selective Iridium(III) complex.

To sensitively detect transcription factor (TF) in complicated biological systems, a ratiometric luminescent detection system based on europium(III) complex-doped silicon nanoparticles (SiNPs) and a G-quadruplex (G4)-selective iridium(III) complex has been successfully constructed. During the biosensor fabrication, the G-rich hairpin DNA H1 was immobilized on the surface of SiNPs which was doped with the europium(III) complex Eu(III)(EDTA)(DPA). The dsDNA (S1/S2) contains TF recognition site, and one of its ssDNA S2 is protected by TF from the digestion of exonuclease III (Exo III). The survived S2 starts a signal amplification process at the aid of Exo III, then a lot of G4 DNA was generated which can be recognized by the G4-selective iridium(III) complex (Ir(III)-2G). So, the emission intensity of Ir(III)-2G is enhanced with the increase of TF concentration, while the emission of Eu(III)(EDTA)(DPA) keeps stable. Ascribed to the long lifetimes of both metal complexes, the constructed detection platform can perform time-resolved emission spectroscopy (TRES) measurement in complicated biological systems. The ratiometric TF detection platform not only can detect NF-κB P50 in buffered system in the linear range of 0.05-5 nM with the limit of detection (LOD) 0.014 nM, but also is able to monitor NF-κB P50 in 10% human serum in the linear range of 0.05-5 nM with the LOD of 0.010 nM through TRES measurement. The two-metal complex-based ratiometric TF biosensor provides new strategy for low concentrations of biomarker detection in the complicated biological systems.

A New Method for Anterior Boundary Demarcation of the Nasopharynx in Three-Dimensional Analysis.

ABSTRACT: Three-dimensional (3D) measurements of the upper airway have been extensively applied and researched, but the division of the airway is carried out in various ways, especially when demarcating the anterior boundary of the nasopharynx. The present study was to propose a new method based on the anatomical definition for the anterior boundary demarcation of the nasopharynx used in three-dimensional analysis. Twenty computed tomography scans (age 9.5 ±â€Š2.5 years, 11 males, and 9 females) of head and neck were randomly selected and transferred to Materialism's interactive medical image control system 19.0 for segmentation of the nasopharynx. Precise localization of the reference points that determining the nasopharyngeal anterior and inferior boundaries was managed by recording their coordinates in the interface of the software. The area of the anterior and the inferior boundaries, and the volume of the nasopharynx were measured and repeated with a 2-week interval for the consistency test. Both the interobserver reliability as well as the intra-observer reliability were very high (intraclass correlation coefficients, 0.985-0.997). Paired t test showed no significant difference between the first and the second examinations. This new simple method proposed for demarcation of the nasopharyngeal anterior boundary was based on the innate anatomical boundary, which was statistically reliable, technically convenient, and clinically reasonable.

What is the impact of distraction osteogenesis on the upper airway of hemifacial microsomia patient with obstructive sleep apnea: a case report.

BACKGROUND: Current research about hemifacial microsomia (HFM) patients after distraction osteogenesis (DO) most emphasize the morphologic changes. This case report shows the outcome of DO on the upper airway of a HFM patient with obstructive sleep apnea (OSA) based on the use of computational fluid dynamics (CFD). CASE PRESENTATION: An 11-year-old boy was diagnosed as HFM with OSA, and underwent unilateral DO. Polysomnography and CT scans were performed before and 6 months after treatment. After DO, lowest blood oxygen saturation increased from 81% to 95% and apnea and hypopnea index decreased from 6.4 events/hour to 1.2 events/hour. The oropharynx and nasopharynx were obviously expanded. We observed apparently increased average pressure, decreased average velocity and pressure drop in all cross-sections, and largely decreased airflow resistance and maximum velocity entirely in the airway. CONCLUSIONS: The results suggest that DO might be effective for the treatment of OSA by expanding the upper airway and reducing the resistance of inspiration.

Morphologic and Aerodynamic Changes of Upper Airway in Pediatric Hemifacial Microsomia Patients Undergoing Distraction Osteogenesis.

Current studies on hemifacial microsomia (HFM) patients undergoing unilateral distraction osteogenesis (DO) mainly confined to description of facial morphology. This study was to investigate the effect of unilateral DO on upper airway in pediatric HFM patients using computational fluid dynamics. The investigators implemented the present retrospective study composed of 20 patients (age 9.5 ±â€Š2.5 years, 11 males, 9 females) with moderate HFM performed unilateral DO on the ramus of mandible. Computational fluid dynamics models of the upper airway were obtained based on the computed tomography data sets which were taken before and 6 months after distraction. Morphologic and aerodynamic parameters were respectively computed and compared at peak inspiration. Paired t-tests were used to compare the differences between the before and after parameters with the significance set at P < 0.05. The authors observed that after DO, oropharynx and nasopharynx were the major expanded regions, average pressure increased in all cross-sections, average velocity decreased significantly except in the superior border of the epiglottis plane, and the airflow resistance significantly reduced in the whole upper airway (P < 0.05). The results indicate that unilateral DO may expand the constricted oropharynx and nasopharynx, obtain a relatively symmetrical airway shape and decrease the airflow resistance which consequently reduce the workload necessary for breathing and facilitate inspiration.

Decontamination of face masks with steam for mask reuse in fighting the pandemic COVID-19: Experimental supports.

The coronavirus disease 2019 pandemic caused by the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has claimed many lives worldwide. Wearing medical masks (MMs) or N95 masks ([N95Ms] namely N95 respirators) can slow the virus spread and reduce the infection risk. Reuse of these masks can minimize waste, protect the environment, and help solve the current imminent shortage of masks. Disinfection of used masks is needed for their reuse with safety, but improper decontamination can damage the blocking structure of masks. In this study, we demonstrated using the avian coronavirus of infectious bronchitis virus to mimic SARS-CoV-2 that MMs and N95Ms retained their blocking efficacy even after being steamed on boiling water for 2 hours. We also demonstrated that three brands of MMs blocked over 99% viruses in aerosols. The avian coronavirus was completely inactivated after being steamed for 5 minutes. Altogether, this study suggested that MMs are adequate for use on most social occasions and both MMs and N95Ms can be reused for a few days with steam decontamination between use.

Potential utilities of mask-wearing and instant hand hygiene for fighting SARS-CoV-2.

The surge of patients in the pandemic of COVID-19 caused by the novel coronavirus SARS-CoV-2 may overwhelm the medical systems of many countries. Mask-wearing and handwashing can slow the spread of the virus, but currently, masks are in shortage in many countries, and timely handwashing is often impossible. In this study, the efficacy of three types of masks and instant hand wiping was evaluated using the avian influenza virus to mock the coronavirus. Virus quantification was performed using real-time reverse transcription-polymerase chain reaction. Previous studies on mask-wearing were reviewed. The results showed that instant hand wiping using a wet towel soaked in water containing 1.00% soap powder, 0.05% active chlorine, or 0.25% active chlorine from sodium hypochlorite removed 98.36%, 96.62%, and 99.98% of the virus from hands, respectively. N95 masks, medical masks, and homemade masks made of four-layer kitchen paper and one-layer cloth could block 99.98%, 97.14%, and 95.15% of the virus in aerosols. Medical mask-wearing which was supported by many studies was opposed by other studies possibly due to erroneous judgment. With these data, we propose the approach of mask-wearing plus instant hand hygiene (MIH) to slow the exponential spread of the virus. This MIH approach has been supported by the experiences of seven countries in fighting against COVID-19. Collectively, a simple approach to slow the exponential spread of SARS-CoV-2 was proposed with the support of experiments, literature review, and control experiences.

A long lifetime ratiometrically luminescent tetracycline nanoprobe based on Ir(III) complex-doped and Eu3+-functionalized silicon nanoparticles.

Different from fluorescent dyes-doped or carbon materials-based ratiometric tetracycline nanoprobes, herein, a new Ir(III) complex-doped and europium(III) ion (Eu3+)-functionalized silicon nanoparticles (Ir(III)@SiNPs-Eu3+) with long luminescent lifetimes was firstly fabricated for selective detection of tetracycline (TC) in complex systems through time-resolved emission spectra (TRES) measurement. In the presence of TC, the red phosphorescence of Eu3+ is greatly enhanced by adsorption energy transfer emission (AETE) of TC, while the strong green luminescence of Ir(III)@SiNPs is quenched by the inner filtration effect (IFE) of TC. Based on these striking emission changes, Ir(III)@SiNPs-Eu3+ can sensitively detect TC in the linear range of 0.01-20 µM with a detection limit of 4.9 × 10-3 µM. Benefitting from the long lifetime of Ir(III)@SiNPs-Eu3+, the nanoprobe demonstrates excellent TC detection performance through TRES in high background system of 5 % human serum. Furthermore, the formed Ir(III)@SiNPs-Eu3+/TC complex can be used to sensitively recognize Hg2+ via a ratiometric luminescence mode. Notably, the cytotoxicity of Ir(III)@SiNPs-Eu3+ is very low and thus the sensitive monitoring the detection of Ir(III)@SiNPs-Eu3+ to TC and Hg2+ also works well in porcine renal cells, demonstrating high application potential in real samples.

γ-Aminobutyric acid-modified graphene oxide as a highly selective and low-toxic fluorescent nanoprobe for relay recognition of copper(II) and cysteine.

A sensitive and selective graphene oxide (GO)-based fluorescent nanoprobe has been developed for the relay recognition of Cu2+ and cysteine (Cys) by covalently grafting γ-aminobutyric acid (GABA) onto GO. The fluorescence of the probe (with excitation/emission maxima at 360/445 nm) is selectively quenched by Cu2+ via static fluorescence quenching. Fluorescence drops linearly as the concentration of Cu2+ is increased from 50 nM to 1.0 µM, and the detection limit for Cu2+ is calculated as 15 nM. By virtue of the strong interaction between Cys and Cu2+, the GO-GABA/Cu2+ complex can further sensitively recognize Cys in a "switch-on" mode. The linear range for Cys detection is from 50 nM to 1.0 µM, and the detection limit is 38 nM. The probe has low cytotoxicity, and it works well inside living cells, which is verified by the successful application in imaging of LLC-PK1 cells. Graphical abstract Gamma-Aminobutyric Acid (GABA) modified graphene oxide (GO) is a highly selective nanoprobe for the fluorometric relay recognition of Cu2+ and Cys.

[Targeted inhibition of Rabies virus gene expression by a chimeric multidomain protein mediated shRNA delivery].

OBJECTIVE: In this study, a new chimeric protein SEG expressed in previous work was applied to evaluate its translocating efficiency of shRNA to rabies virus infected cells in mice, meanwhile, the capability of anti-rabies virus was investigated. METHODS: Rabies virus strain CVS-24 was inoculated into the hind leg to establish a mouse model of rabies in a dose of 50 LD50; 12 h thereafter the mice were injected intravenously with shRNA-producing plasmid mixed with SEG. To test shRNA delivery, single-cell suspensions from brain, spleen and liver were examined by flow cytometry. Rabies virus in brain tissue of mice was detected by qRT-PCR, RT-PCR, western blot and directed immunofluorescence assay. Mice were monitored for survival and serum samples were tested for IFN-α levels. RESULTS: No green fluorescent protein (GFP) was seen in the spleen or liver, suggesting that SEG allows specific targeting of RV-infected cells. RT-PCR and western blot showed that mice treated with SEG-shRNA had lower rabies virus RNA and protein levels than the controls. Real-time PCR showed that rabies virus was reduced 4.88 fold compared to the mock cells. Survival of RV-infected mouse showed a significant protection from rabies virus infection by SEG-shRNA treatment. The survival was up to 50% whereas the control group all died. IFN was not induced in SEG-shRNA treated animals. CONCLUSION: shRNA-producing plasmid was specifically delivered into rabies virus infected cells using the SEG protein, and effectively inhibited rabies virus geneexpression and replication in vivo. SEG-shRNA can be used for adjuvant treatment for rabies.

WITHDRAWN: Development of an RT-PCR-RFLP assay for the detection and differentiation of wild-type and vaccine strains of classical swine fever virus.

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[Construction and detection the bioactivity of human anti-rabies virus scdsFv].

OBJECTIVE: The purpose of this study is to produce new antibody molecules to neutralize the rabies virus specifically by the way of constructing and expressing the human anti-rabies virus scdsFv (disulfide stabilized single chain antibody) gene, and characterizing its bioactivity. METHODS: We obtained the sequence of variable region of heavy chain (VH) and variable region of light chain (VL) of RV monoclonal antibody SO57 from GenBank, and it respectively mutated into cysteine in the gene loci VH44 and VL100. The scdsFv gene was synthesized and inserted into a prokaryotic expression vector pET22b(+). Purified inclusion body scdsFv proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The binding activity of scdsFv was identified by ELISA and mouse brain tissues infected with rabies virus CVS strain. The relative affinity of scdsFv was measured by ELISA using thiocyanate elution. The capacity of the scdsFv to neutralize the rabies virus CVS strains was determined by fluorescent antibody virus neutralisation test (FAVN). The fusion proteins neutralized the CVS strain in a standard in vivo neutralized assay where the virus was incubated with the scdsFv molecules before intracranial inoculation in mice. RESULTS: The fragment genes of scdsFv to rabies virus were constructed successfully. ScdsFv protein was expressed in E. coli with approximate molecular weight of 30.0 kDa, which could be recognized by anti-His mAb. ELISA results demonstrated that scdsFv could bind antigen specificity. It was found that the strong reactivity of scdsFv to the smear of RV infected mouse-brain was demonstrated by IFA. As determined by FAVN with a reference serum, the titer of scdsFv was 41 IU/mL. In addition, scdsFv could be 55.6% protection of mice against lethal challenge with rabies virus CVS. CONCLUSION: The scdsFv can bind antigen specificity and has neutralization capacity to the virus in vitro and in vivo. The anti-rabies scdsFv is potential for application in rabies post-exposure prophylaxis.

A rapid immunochromatographic test strip for detecting rabies virus antibody.

An immunochromatographic test strip (ICTS) for detecting antibodies to rabies virus was developed, using colloidal gold particles labeled with rabies virus glycoprotein as the tracer. The assay was evaluated using sera from dogs immunized with various commercial rabies vaccines, or from dogs in the clinics and sera from dogs immunized with vaccines against pathogens other than rabies virus, and negative sera from a wide variety of animal sources, including dogs, mice, and cats which had never been vaccinated. The ICTS was found to be highly specific for antibodies against rabies virus, with a detection limit of 0.5IU/ml as measured by the fluorescent antibody virus neutralization (FAVN) test. Compared with the FAVN test, the specificity and sensitivity of ICTS were 98.2% and 90.4%, respectively. There was an excellent agreement between results obtained by the ICTS and FAVN tests (kappa=0.888). Strips stored at 4°C in a plastic bag with a desiccant retained their specificity and sensitivity for at least 15 months, and strips stored at ambient temperature remained stable for 12 months. The immunochromatographic test strip may therefore be useful for clinical laboratories lacking specialized equipment and for diagnosis in the field for rapid detection of rabies virus-specific antibodies.

[Targeted inhibition of rabies virus replication in vitro by single chain antibody domain mediated vector expression shRNA delivery].

OBJECTIVE: Single chain antibody-mediated delivery is a novel approach for targeting shRNA to appropriate cells. In this report, we studied whether this shRNA delivery strategy would be effective against rabies virus. METHODS: Rabies virus scFv (G) gene and ETA-GAL4 gene were amplified by PCR from vector scFv (G)-T and PE40-GAL4-T respectively. Then, the chimeric gene scFv (G)-ETA-GAL4 was constructed by lapextension PCR and cloned into the prokaryotic expression vector pET28a( +). Recombinant expression plasmid of pET28a(+)-scFv(G)-ETA-GAL4 was constructed and then transformed into the competent E. coli BL21 (DE3) cells for expression under the induction of IPTG. scFv(G)-ETA-GAL4 protein was purified by Ni-NTA His Bind Resin affinity chromatograph and identified by SDS-PAGE gel and Western blot assay. Binding of the fusion protein scFv(G)-ETA-GAL4 to rabies virus was determined by ELISA. Complexes which formed spontaneously by the fusion protein scFv(G)-ETA-GAL4 with plasmid pRNATU6.3-shRNA were added to BHK-21 cells culture medium that infected with RV. Green fluorescent protein (GFP) was observed after 35 h and judged the transferring efficiency of the complexes. The inhibition of RV replication by shRNA was detected by direct immune fluorescence test. RESULTS: A 1557 bp DNA encoding scFv (G)-ETA-GAL4 protein gene was cloned and successfully expressed in inclusion body with approximate molecular weight of 57.0 kDa, which could be recognized by anti-His mAb. The scFv(G)-ETA-GAL4 proteins were purified by Ni-NTA column , and after renatured with the yield of 2.8 mg/mL. The ELISA results showed that when concentration of the scFv(G)-ETA-GAL4 protein ranging from 2.8 nmol/L to 1000 nmol/L, binding affinity is directly related with RV. The GFP expressed in BHK-21 cell after transfection with the complexes and effectively inhibited RV replication in BHK-21 cell. CONCLUSION: scFv(G)-ETA-GAL4 fusion protein could mediated plasmid pRNATU6.3-shRNA transferred into BHK-21 cell infected with RV, and then inhibited RV replication.