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.

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.

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×10^9.1 TCID₅₀/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.
Adenoviridae/genetics* ; Adenoviruses, Human/genetics* ; Animals ; Antibodies, Viral ; Capsid/metabolism* ; Capsid Proteins ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins/genetics* ; Serogroup ; Swine ; Viral Proteins ; Viral Vaccines/genetics*

Many people affected by fragile X syndrome (FXS) and autism spectrum disorders have sensory processing deficits, such as hypersensitivity to auditory, tactile, and visual stimuli. Like FXS in humans, loss of Fmr1 in rodents also cause sensory, behavioral, and cognitive deficits. However, the neural mechanisms underlying sensory impairment, especially vision impairment, remain unclear. It remains elusive whether the visual processing deficits originate from corrupted inputs, impaired perception in the primary sensory cortex, or altered integration in the higher cortex, and there is no effective treatment. In this study, we used a genetic knockout mouse model (Fmr1^KO), in vivo imaging, and behavioral measurements to show that the loss of Fmr1 impaired signal processing in the primary visual cortex (V1). Specifically, Fmr1^KO mice showed enhanced responses to low-intensity stimuli but normal responses to high-intensity stimuli. This abnormality was accompanied by enhancements in local network connectivity in V1 microcircuits and increased dendritic complexity of V1 neurons. These effects were ameliorated by the acute application of GABA_A receptor activators, which enhanced the activity of inhibitory neurons, or by reintroducing Fmr1 gene expression in knockout V1 neurons in both juvenile and young-adult mice. Overall, V1 plays an important role in the visual abnormalities of Fmr1^KO mice and it could be possible to rescue the sensory disturbances in developed FXS and autism patients.
Animals ; Disease Models, Animal ; Fragile X Mental Retardation Protein/metabolism* ; Fragile X Syndrome/metabolism* ; Humans ; Mice ; Mice, Knockout ; Neurons/metabolism*

Objective To demonstrate that MRI is more advantageous than ultrasound in the detection of ischemic cerebral lesions precisely in twin-twin transfusion syndrome(TTTS)fetus following fetoscopic selective laser coagulation(SLCPV).Methods The fetus with TTTS treated by SLCPV were collected.All fetuses underwent brain MRI within 1-5 days after the procedure and a follow-up MRI at 29-32 weeks'gestation.All fetuses also had frequent ultrasound evaluation until delivery.Results Twenty-five pregnancies with TTTS were included in the study.Six pregnancies resulted in survival of only one twin after the procedure(Group A),while the other 19 pregnancies resulted in the survival of both twins(Group B),as assessed at the time of initial MRI.Two fetus in Group A and three in Group B had evidence of bleeding in lateral ventricle.Two fetus in Group B had actue brain ischemia.One fetus in Group B had appeared brain atrophy and choosed to terminate pregnancy.Conclusion MRI can make a clearer diagnosis of cerebral damage of TTTS fetus in a short period after SLCPV.

Objective There have been many techniques proposed for the reconstruction of pancreatic digestive continuity to prevent fistula (PF) formation, but this is still highly debated. We carried out a systematic review and meta- analysis to determine the effectiveness of methods of anastomosis after pancreaticoduodenectomy (PD). Methods A full literature search was conducted in the Cochrane Controlled Trials Register Databases, Medline, et al.Randomized controlled trials(RCTs) were considered for inclusion. Analysis was carried out using Revman software. Results In all, 10 RCTs including a total of 1 408 patients were included, the pancreaticogastrostomy (PG) and pancreaticojejunostomy (PJ) groups, duct to mucosa PJ and PJ, binding PJ and PJ, pancreatic duct without anastomosis PJ and PJ. The meta-analysis showed that the PF, postoperative complications, biliary fistula, mortality, re-operation and hospital stay were not statistically different among four methods(P>0.05).Conclusions No ideal technique of pancreatic reconstruction after PD is found to be applicable to all kinds of pancreatic remnants in this systematic review and meta-analysis.

Object To study the expression of miRNA-146a in sepsis-induced acute lung injury (ALI) patients and its effect on the inflammation in mouse models.Methods miRNA-146a expression in peripheral blood was determined in sepsisinduced ALI patients and healthy volunteers by qRT-PCR.Sepsis-induced ALI mouse model were reproduced by LPS treatment and miRNA-146a mimic,miRNA-146a NC and miRNA-146a inhibitors were injected through trachea.The expressions of miRNA-146a,TNF-α,IL-1β and COX-2 mRNA were determined by qRT-PCR 24h after the intervention.Results The miRNA-146a expression in peripheral blood significantly increased in severe sepsis with ALI patients,compared with the control subjects.For mouse model,the expressions of TNF-o,IL-1β and COX-2 mRNA significantly decreased in miRNA-146a group,while increased in miRNA-146a inhibitor group compared with miRNA-146a NC group (P＜0.05).The TNF-α and IL-1β expressions significantly decreased in miRNA-146a inhibitor+COX-2 inhibitor group compared with miRNA-146a inhibitor group (P＜0.05).Conclusion MiR-146a treatment can effectively alleviate the lung inflammation in sepsis-induced ALI mice.

Purpose To discuss the value of MRI in the diagnosis of placenta implantation abnormality, and to explore preliminarily the relationship between MRI signs and types of placenta implantation abnormality. Materials and Methods The clinical preoperative data and postnatal pathological findings of 54 women at high risk of placenta accreta were collected. All the patients undertook the conventional pelvic MRI examination. The scanning sequences mainly included: sagittal, coronal and axial T2-weighted imaging-turbo spin echo, balance fast field echo. The MRI images were observed and the areas which showed low signal in all the three directions on T2WI were measured. Then the correlation between the areas of low signal on T2WI in placental and the types of placenta implantation abnormality was analyzed. Results The incidence of placenta implantation abnormality was 64.8% in our research (35/54). The main MRI signs were low signal on T2WI (68.5%, 37/54) and heterogeneous signal in placenta (57.4%, 31/54); the main sign of placenta percreta was tenting bladder (75.0%, 6/8). The types of placenta implantation abnormality were positively correlated with the areas of low signal on T2WI (r=0.454, P<0.05). Conclusion Pregnant women at risk of placenta accreta should be evaluated with imaging examinations, particularly with MRI scanning, to improve disease detection rate. The typical indirect signs of placenta implantation abnormality are low signal on T2WI and heterogeneous signal in placenta. The larger size of low signal area on T2WI in placenta, the deeper implantation of placenta.