This study aims to establish an antibody detection method for porcine deltacoronavirus (PDCoV). The recombinant proteins PDCoV-N1 and PDCoV-N2 were expressed via the prokaryotic plasmid pColdII harboring the N gene sequence of the PDCoV strain CH/XJYN/2016. The reactivity and specificity of PDCoV-N1 and PDCoV-N2 with anti-PEDV sera were analyzed after the recombinant proteins were analyzed by SDS-PAGE and purified by the Ni-NTA Superflow Cartridge. Meanwhile, Western blotting and indirect immunofluorescence assay were carried out separately to validate the recombinant proteins PDCoV-N1 and PDCoV-N2. Finally, we established an indirect ELISA method based on the recombinant protein PDCoV-N2 after optimizing the conditions and tested the sensitivity, specificity, and reproducibility of the method. Then, the established method was employed to examine 102 clinical serum samples. The recombinant protein PDCoV-N2 showed low cross-reactivity with anti-PEDV sera. The optimal conditions of the indirect ELISA method based on PDCoV-N2 were as follows: the antigen coating concentration of 1.25 μg/mL and coating at 37 ℃ for 1 h; blocking by BSA overnight at 4 ℃; serum sample dilution at 1:50 and incubation at 37 ℃ for 1 h; secondary antibody dilution at 1:80 000 and incubation at 37 ℃ for 1 h; color development with TMB chromogenic solution at 37 ℃ for 10 min. The S/P value ≥ 0.45, ≤0.38, and between 0.45 and 0.38 indicated that the test sample was positive, negative, and suspicious, respectively. The testing results of the antisera against porcine epidemic diarrhea virus (PEDV), porcine circovirus 2 (PCV2), transmissible gastroenteritis virus (TGEV), foot-and-mouth disease virus (FMDV), and African swine fever virus (ASFV) showed that the S/P values were all less than 0.38. The testing results of the 800-fold diluted anti-PDCoV sera were still positive. The results of the inter- and intra-batch tests showed that the coefficients of variation of this method were less than 10%. Clinical serum sample test results showed the coincidence rate between this method and neutralization test was 94.12%. In this study, an ELISA method for the detection of anti-PDCoV antibodies was successfully established based on the truncated N protein of PDCoV. This method is sensitive, specific, stable, and reproducible, serving as a new method for the clinical diagnosis of PDCoV.
Animals ; Enzyme-Linked Immunosorbent Assay/methods* ; Swine ; Antibodies, Viral/blood* ; Recombinant Proteins/genetics* ; Deltacoronavirus/isolation & purification* ; Coronavirus Infections/virology* ; Swine Diseases/diagnosis* ; Coronavirus Nucleocapsid Proteins ; Sensitivity and Specificity

Feces and intestinal contents of pigs suspected with porcine epidemic diarrhea virus were collected from a farm in Minqin County,Gansu Province,China.After the suspected positive sam-ples were detected by RT-PCR,Vero cells were used to isolate and culture them in vitro.The suc-cessfully isolated virus was identified in the laboratory,and its whole genome sequence was ana-lyzed for genetic evolution.The pathogenicity was evaluated by animal regression test.The results showed that typical syncytial lesions could be observed when the PEDV-positive treatment solu-tion was inoculated with Vero cells in the 4th generation,and the virus titer in the 6th generation reached 10-4 75TCID50/mL.PEDV-like virions with a diameter of about 100 nm and a round shape with obvious capsular membranes and spikes were observed by electron microscopy.Whole genome sequencing analysis showed that the total length of this strain was 28 085 bp,which was far from the G1 subtype represented by the classical strain CV777(96.6％),and had a high homology with the G2b strains BC-2011-1,IA1,USA/Colorado/2013 and WELL(98.6％).This indicated that the strain belonged to the G2b epidemic strain.The animal regression test showed that the 5-day-old piglets developed vomiting,acute watery diarrhea,emaciation and mental depression within 12 h after the attack,and the symptoms worsened and died within 24 h.After autopsy,the infected piglets could be observed with stomach swelling,high intestinal heave,thin and transparent intesti-nal wall,and undigested milk clots inside.In summary,a PEDV G2b epidemic strain was success-fully isolated and identified in this study,and its whole genome sequence and pathogenicity were analyzed,providing research materials for future studies on PEDV gene function,pathogenic mech-anism and vaccine development.

Food allergy(FA)is a pathological and potentially fatal immune response,whose incidence continues to rise and has become an important public health problem affecting children and adults.Short chain fatty acids(SCFAs),produced by bacteria,through the fermentation of dietary fibre in the gut,played an important role in in pathogenesis of FA.In this review,we will discuss the immunomodulatory effects of SCFAs on the intestinal epithelial cells and various immune cells and their related molecular mecha-nisms in the context of FA,providing new perspectives for the prevention and treatment of FA.

To screen and identify the key host proteins interacting with the non-structural protein 15 (Nsp15) of porcine epidemic diarrhea virus (PEDV). The IP/pull-down assay and mass spectrometry were employed to screen and identify the host proteins interacting with Nsp15. The interaction between the host protein and Nsp15 was studied by co-immunoprecipitation and laser scanning confocal microscopy. Finally, Western blotting and RT-qPCR were employed to examine the interaction between SLC25a3 and PEDV. The recombinant eukaryotic expression vector pcDNA3.1(+)-Flag-Nsp15 was successfully constructed, and the host protein SLC25a3 interacting with PEDV Nsp15 was screened out. An interaction existed between SLC25a3 and Nsp15, and SLC25a3 significantly inhibited PEDV replication in a dose-dependent manner. SLC25a3 inhibits PEDV replication. The results of this study provide a basis for deciphering the role and mechanism of SLC25a3 in the host immune response to PEDV infection.
Porcine epidemic diarrhea virus/genetics* ; Viral Nonstructural Proteins/metabolism* ; Animals ; Swine ; Virus Replication ; Coronavirus Infections/veterinary* ; Swine Diseases/metabolism*

Improving the reproducibility of biomedical research results is a major challenge.Transparent and accurate reporting of the research process enables readers to evaluate the reliability of the research results and further explore the experiment by repeating it or building upon its findings. The ARRIVE 2.0 guidelines, released in 2019 by the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), provide a checklist applicable to any in vivo animal research report. These guidelines aim to improve the standardization of experimental design, implementation, and reporting, as well as the reliability, repeatability, and clinical translatability of animal experimental results. The use of ARRIVE 2.0 guidelines not only enriches the details of animal experimental research reports, ensuring that information on animal experimental results is fully evaluated and utilized, but also enables readers to understand the content expressed by the author accurately and clearly, promoting the transparency and integrity of the fundamental research review process. At present, the ARRIVE 2.0 guidelines have been widely adopted by international biomedical journals. This article is a Chinese translation based on the best practices of international journals following the ARRIVE 2.0 guidelines in international journals, specifically for the complete interpretation of the ARRIVE 2.0 guidelines published in the PLoS Biology journal in 2020 (original text can be found at https://arriveguidelines.org). The fourth part of the article includes the items 1-5 of ARRIVE 2.0 Recommended 11 section, which covers "Abstract" "Background" "Objectives" "Ethical statement" and "Housing and husbandry". Its aim is to promote the full understanding and use of the ARRIVE 2.0 guidelines by domestic researchers, enhance the standardization of experimental animal research and reporting, and promote the high-quality development of experimental animal technology and comparative medicine research in China.

Objective To investigate the protective effects on the renal allografts from brain dead (BD) donor rats pretreated with bone marrow mesenchymal stem cells (MSCs).Method Three groups [normal transplant group (G1).BD transplant group (G2),and MSCs pretreated + BD transplant group (G3)] were set up.Male F344 rats served as donors and male Lewis rats as recipients.In G1,kidneys from F344 donor rats were implanted into Lewis recipients.In G2,kidneys from F344 BD donor rats were engrafted into Lewis recipients.In G3,after BD was established in F344 rats,MSCs were given intravenously to the rats.The kidneys harvested 6 h later were transplanted to Lewis recipients.Cyclosporine was intromuscularly given daily to the recipient rats for 10 days.Right kidneys were resected from recipients on day 10.Creatinine level was examined on day 14,21,28,and 35.Renal allografts harvested on day 35 were pathologically detected.The irnmunochemistry expression of interleukin (IL)-1β and tumor necrotic factor (TNF)-α in renal allograft tissue was tested.Result Serum creatinine levels in G2 were remarkably higher than those in G1 and G3 (P＜0.01) on day 14,21,28,and 35 postoperatively.The creatinine levels on the above mentioned time points had no statistically significant difference between G3 and G1 except on day 21.Postoperative pathological changes in G2 of both pronounced infiltration of mononuclear cells and tubular epithelia[inflammation were notably increased in renal allografts as compared with those in G1 and G3.There was no obvious difference between G1 and G3 in infiltrated mononuclear cells and tubular epithelial inflammation.Positive expression levels of both IL-1β and TNF-α in glomerular,tubular and interstitial epithelial cells were statistically enhanced in G2 as compared with those in G1 and G3 (H =7.210,P =0.027),while there was no statistically significant difference in the expression of both IL-1[β and TNF-α between G1 and G3.Conclusion Brain dead donor rats pretreated with bone marrow MSCs might reduce renal allograft injury via decreasing both inflammatory cell infiltration and IL-1β and TNF-α expression.

Objective In this article Response Surface Analysis(RSA)was applied to optimize the formulation of doxycycline hydrochloride sustained release tablet.Methods Single factor exploration was used to determine the three factors which have the greatest impact on the release rate.The three factors were the dosage of the HPMC in the total weight of the tablet,the concentration of PVP-K30,and the ratio of lactose to microcrystalline cellulose,respectively.The composite score of the release behaviour was taken as the response value.The dosage of the ingredients were determined by Box-Benhnke design principles and 3 factors and 3 levels.Results The optimized formulation and process are as follows:the dosage of the HPMC in the total weight of the tablet was 30%;the concentration of PVP-K30 was 10%,and the ratio of lactose to microcrystalline cellulose was 13.The release behavior in vitro is ideal.Conclusion The optimized preparation process of doxycycline hydrochloride sustained release tablet is stable,highly efficient and suitable for industrial production.

To prepare the polyclonal antibody against the 3D polymerase of foot and mouth disease virus (FMDV), the 3D polymerase gene of this virus was amplified by PCR and doubly digested with BamH I and Nde I. Then, it were cloned into expression vector pET-30a(+) to obtain the recombinant plasmid pET-3D and this plasmid was transformed to E.coli BL21(DE3) with induction by IPTG.The target protein was identified and purified with SDS+PAGE, and the inclusion bodies were extracted. The purified target protein was used as antigen to immunize New Zealand rabbits to prepare the polyclonal antibody against 3D polymerase of FMDV, which was then characterized by indirect ELISA and Western blotting. As demonstrated by SDS-PAGE, the target protein with a molecular weight at 46 ku was expressed. The polyclonal antibody showed high affinity and obvious specificity and its titer was above 1:8 000. This polyclonal antibody may lay a foundation for the further studies on the biological functions and epitopes of the 3D polymerase of FMDV.

ObjectiveTo investigate the relationship between the prognosis and both of treatment way and curative age of congenital muscular torticollis.Methods138 children accepted conservative and/or operative treatment according to different ages.A prospective study had been made. ResultsConservative treatment was given to 82 children,71 of them were <1-year-old. 49 of them were follwed up and had satisfactory outcome.7 patients who was >1-year-old had operations later because of asymmetry of head.63 children received operations.56 of them were followed up over one year,47 cases had good-looking,the outcome of other 9 was not satisfied.Conclusions Operative intervention was necessary for >1-year-old patients. Manipulation was suitable way to cure <1-year-old patients.It was the best operating time at the age of 1-5 years old.