The highly virulent PRRSV isolate strain HN-1/06 was cultivated on Marc-145. To study the viral entry mechanisms, the GP5 gene of PRRSV isolate was amplified by RT-PCR and cloned into pcDNA3.0 to generate the expressing plasmid pcDNA-GP5. pcDNA-GP5 was transfected into 293T by the calcium phosphate precipitation method. Analysis of flow cytometry confirmed that the GP5 proteins were expressed in surface of the 293T cells. Then 293T cells were transfected with pcDNA-GP5, pHIT60 and pHIT111 plasmids to generate pseudotyping virus. The pseudotyping virus supernatant was harvested 48 hours post-transfection and was detected by Western blotting and infection assay. Western blotting indicated that the GP5 glycoproteins were incorporated into the retroviral pseudotyped virus. Infection assay showed that the pseudotyped virus infected 293T and Mark-145 cell. The pseudotyped virus could be used to further study infectious mechanism of PRRSV.
Animals ; Cell Line ; Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; virology ; Leukemia Virus, Murine ; genetics ; metabolism ; Mice ; Porcine respiratory and reproductive syndrome virus ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; Virion ; genetics ; metabolism

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing viral encephalitis in humans and reproductive disorder in swine. JEV is prevalent throughout China in human; however, spatiotemporal analysis of JEV in Chinese swine herds has not been reported previously. Herein, we present serological and molecular epidemiological results and estimates of prevalence of JEV infections among swine herds in various regions of China. The results suggest that JEV infections are widespread and genotype I and III strains co-exist in the same regions. Therefore, there is an urgent need to monitor JEV infection status among swine herds in China.
Asian Continental Ancestry Group ; China ; Encephalitis Virus, Japanese ; Encephalitis, Japanese ; Encephalitis, Viral ; Flavivirus ; Genotype ; Humans ; Molecular Epidemiology ; Prevalence ; Spatio-Temporal Analysis ; Swine

【Objective】 To investigate the factors affecting the length of hospitalization after the Sun's procedure in patients with type A aortic coarctation. 【Methods】 From January 2018 to June 2023, the clinical data, related laboratory indicators and perioperative blood transfusion data of patients with type A aortic dissection who underwent Sun's procedure in the First Hospital of Lanzhou University were collected. LASSO regression was used to screen the characteristics related to the length of hospital stay, and linear regression analysis was used to determine the risk factors for prolonged length of hospital stay. 【Results】 The statistical analysis of 242 patients showed that the amount of red blood cell transfusion, plasma transfusion, platelet transfusion and autologous blood transfusion were the influencing factors of the length of hospital stay in patients with type A aortic dissection after operation. The total sum of squared deviations of the linear regression equation fitting the total length of hospital stay was statistically significant (F= 10.504, P<0.001). 【Conclusion】 Perioperative red blood cell transfusion,plasma transfusion,platelet transfusion and autologous blood transfusion are risk factors for prolonged postoperative hospitalization in patients undergoing the Sun's procedure for type A aortic coarctation. Control of operation time and reduction of intraoperative blood loss may help to prevent prolonged postoperative hospital stay and other adverse conditions.

【Objective】 To investigate the serology and genotype identification method of B (A) subtype patients. 【Methods】 Test tube method (serology) was used to confirm the clinically difficult ABO blood group samples of 3 patients with ABO blood group; ABO blood group was genotyped by real-time PCR, and the ABO gene exon 1-7 was sequenced to determine the genotype. 【Results】 The forward and reverse blood typing result of three patients was B (A) subtype all with ABO genotype B/O2 and c.640A> G mutation on B allele of exon 7, which meets the characteristics of ABO * BA.04 genotype. 【Conclusion】 The combination of serological and genetic testing could identify difficult blood types such as ABO subtypes accurately and ensure the safety of clinical blood use.

Objective:To prepare and identify a broad-spectrum antibody FHA3 targeting influenza A virus hemagglutinin (HA).Methods:According to the single-chain antibody fragment (scFv) sequence, the heavy chain (VH) and light chain (VL) variable regions of FHA3 were amplified by PCR and a recombinant plasmid pFRT-IgG1κ-FHA3 was constructed by linking the expression vector pFRT-IgG1κ. FHA3 was expressed in the ExpiCHO system and purified by affinity purification. The binding activity of FHA3 to influenza A virus HA was detected by ELISA. The neutralizing activity of FHA3 was detected in vitro by infecting host cells with pseudovirus. Results:SDS-PAGE showed that high-purity FHA3 was obtained. FHA3 could bind to H1N1 HA, H2N2 HA, H3N2 HA, H5N1 HA, H7N9 HA and H9N2 HA in a concentration-dependent manner. FHA3 had good neutralizing activity in vitro that was it could effectively block the invasion of H5N1 and H7N9 pseudoviruses into target cells at a low concentration of 5 μg/ml and H1N1 pseudovirus at 0.012 5 μg/ml. Conclusions:A broad spectrum antibody targeting HA protein of influenza A virus with neutralizing activity in vitro was obtained.

Objective:To prepare and identify a functional antibody FNA1 targeting the neuraminidase (NA) of influenza A virus N1 subtype.Methods:According to single-chain antibody fragment (scFv) sequence, the heavy chain and light chain variable region sequences of FNA1 were synthesized, and the recombinant expression plasmid pFRT-IgG1κ-FNA1 was constructed by linking the expression vector pFRT-IgG1κ. The FNA1 antibody was expressed in ExpiCHO cells and purified using affinity purification technique. The binding ability of FNA1 to the target proteins, influenza A virus N1 subtype NA antigens, was detected by ELISA. Flow cytometry was performed to analyze the binding ability of FNA1 to the NA antigens expressed on the surface of cell membrane. The in vitro activity of FNA1 against NA was evaluated by infecting 293T cells with pseudovirus. Results:Protein electrophoresis showed that FNA1 with high purity was obtained. FNA1 specifically recognized and bound to N1 subtype NA antigens in a concentration-dependent manner. FNA1 could effectively block NA activity by binding to N1 subtype NA protein expressed on the surface of cell membrane, thus inhibiting the release of packaged pseudovirus from cell surface and further inhibiting target cell infection.Conclusions:An antibody FNA1 targeting influenza A virus N1 subtype NA with in vitro functional activity was obtained.

Objective:To establish an antibody expression system to reduce the antibody-dependent enhancement (ADE) effect of target antibody.Methods:Site-directed mutagenesis was used to mutate the 234 and 235 sites of the Fc region of the mammalian cell antibody expression vector-L234A and L235A to establish the antibody expression vector pFRT-IgG1κ-FcM. An antibody Wt-WNV with significant ADE effect obtained in previous work was selected and expressed by the pFRT-IgG1κ-FcM system to obtain mutant antibody FcM-WNV. The binding ability of FcM-WNV to target antigen West Nile virus envelope protein-DⅢ (WNV E-DⅢ) was detected by ELISA, and the its binding ability to human high-affinity IgG Fc receptor hFcγRⅠ (hCD64 ) was analyzed by flow cytometry. The neutralizing activity of FcM-WNV in vitro was detected by pseudovirus infection of host cells (BHK21 and K562). Results:The expression levels of FcM-WNV and Wt-WNV were comparable, and FcM-WNV could recognize and bind to WNVE-DIII in a concentration-dependent manner. Compared with Wt-WNV, the binding ability of FcM-WNV to hCD64 was significantly weakened, showing a significant decrease in fluorescence intensity. Consistent with the previous experimental results, Wt-WNV at a concentration of 5 μg/ml significantly enhanced the infection of K562 by WNV pseudovirus, while FcM-WNV at a concentration of 5 μg/ml could effectively block pseudovirus infection in both K562 and BHK21 cells.Conclusions:The established antibody expression system can effectively reduce the ADE effect of the target antibody.

Objective:To establish an in vivo infection model of H5N1 pseudovirus and to detect the neutralizing activity of FHA3 antibody using this model. Methods:Based on the sequence information of hemagglutinin (HA) and neuraminidase (NA) of A/Anhui/1/2005/H5N1 strain, two recombinant plasmids of pcDNA3.1-HA5 and pcDNA3.1-NA1 were constructed. The two plasmids and plasmid pNL4-3.Luc.R-E- were co-transfected into 293T cells to prepare H5N1 pseudovirus supernatant. The morphology of pseudovirus particles in the supernatant was observed by electron microscopy. MDCK cells were infected with the pseudovirus supernatant and the virus titer was detected. BALB/c mice were injected with the pseudovirus supernatant by intraperitoneal injection and subjected to bioluminescence imaging at 2, 5, 8, and 12 d after infection to detect the pseudovirus infection in vivo. The functional activity of FHA3 antibody in vivo was evaluated using the established mouse infection model. Results:The recombinant plasmids pcDNA3.1-HA5 and pcDNA3.1-NA1 were correctly constructed and could be used to prepare pseudovirus supernatants of high titer by co-transfecting 293T cells with the plasmid pNL4-3.Luc.R-E-. The virus particles were round under electron microscope. H5N1 pseudovirus-infected mice exhibits strong fluorescence signals, which were attenuated by FHA3 treatment before challenge.Conclusions:The in vivo infection model of H5N1 pseudovirus was successfully constructed and FHA3 antibody was proved to be protective against the pseudovirus infection.

Objective: To investigate the effects of XIAOJI Decoction combined with FOLFOX chemotherapy on serum cytokine expression profile in patients with advanced colorectal carcinoma by liquid chip technology. Methods: Fourteen patients with advanced colorectal carcinoma, who met the inclusion criteria and were treated in the Department of Oncology, Higher Education Mega Center Hospital of Guangdong Provincial Hospital of Traditional Chinese Medicine during January 1, 2018 and December 31, 2018 were retrospectively analyzed in this study. The patients were divided into chemotherapy group (n=7, treated with 5-Fluorouracil + Calcium Folic Acid+Oxaliplatin (FOLFOX)) and combined treatment group (n=7, treated with XIAOJI Decoction + FOLFOX) according to therapeutic scheme. The curative efficacy was evaluated after 6 treatment courses. The expression profile of cytokines in blood serum of patients was examined by liquid chip technology after every 2 courses. Results: Fourteen patients received a total of 84 cycles of therapy. Survival analyses showed that the progress-free survival time (PFS) and overall survival time (OS) of two groups couldn't be compared due to insufficient samples, although the combined treatment group had longer PFS (10 months vs 6 months) and OS (17 months vs 12 months) than the chemotherapy group.As to adverse reactions, the rates of leucopenia, diarrhea, nausea, peripheral neuritis and alopecia in two groups were comparable, while the severity in combined treatment group were lighter than that in chemotherapy group. In comparison with the combined treatment group, concentrations of serum BDNF and IL-2 were statistically higher in the chemotherapy group (P<0.05). By comparing the cytokine concentrations at different collection time points before and after the treatment, it showed that the concentration of serum IL-2 in chemotherapy group was higher than that in combined treatment group after 2 courses of treatment (P<0.05). In total, there were 19 cytokines showed a tendency to be higher in combined treatment group than chemotherapy group during different treatment periods. Conclusion: Combined treatment of XIAOJI Decoction with FOLFOX for advanced colorectal carcinoma is a treatment option worth exploring, and liquid chip analysis showed that the mechanism may be related to the reduction of serum LI-2 and BDNF levels in patients.

Objective To investigate the mechanism and clinical significance of miR-18a-5p and retinoid acid receptor-related orphan receptor-α (RORA) in the proliferation and progression of colorectal cancer (CRC) cells. Methods The expressions of miR-18a-5p and RORA in CRC cells and tissues were detected via qRT-PCR, FISH, and IHC. Cell proliferation capability was detected through EdU and CFSE assay, cell apoptosis by flow cytometry assay, and cell migration and invasion abilities by cell scratch and Transwell invasion assays, respectively. The targeted regulation of miR-18a-5p on RORA was further verified via dual-luciferase reporter assay, cell function rescue test, RT-PCR, and Western blot assay. Finally, bioinformatics was used to explore the molecular mechanism of miR-18a-5p promoting malignant proliferation, invasion, and progression of CRC via regulating RORA. Results miR-18a-5p exhibited a high expression in CRC tissues and cells (P<0.05) and promoted the proliferation, migration, and invasion of CRC cells (P<0.05). In addition, RORA served as the target gene of miR-18a-5p, and its overexpression effectively reduced the promoting function of miR-18a-5p in the malignant biological phenotype of CRC cells (P<0.05). The expression of RORA in CRC tissues showed a significantly positively correlation with the infiltration of CD8+T cells and the expression of its surface marker protein CD8A. Conclusion The targeted regulation of RORA by miR-18a-5p promotes the proliferation and progression of CRC. The miR-18a-5p/RORA regulatory pathway possibly contributes to the immune microenvironment of CRC, which can be a potential therapeutic target for CRC.