Objective:To analyze the protective effect of a respiratory syncytial virus (RSV) vaccine based on bacterial outer membrane vesicle (OMV) in mice.Methods:The pre-fusion protein (preF) of RSV was linked to the surface of OMV through the transmembrane protein cytolysin A (ClyA) to form the nanovaccine OMV-preF. The morphological characteristics of OMV and OMV-preF were observed under a transmission electron microscope. OMV-preF was intramuscularly injected into BALB/c mice and the elicited humoral and cellular immune responses were evaluated. The protective effect of OMV-preF was assessed by challenging the immunized mice with RSV Long strain. One-way analysis of variance and Tukey test were used for statistical analysis.Results:The results showed that preF was stably expressed in OMV, and both OMV-preF and OMV exhibited a double-layer vesicle structures under the microscope. OMV-preF could significantly activate the cellular and humoral immune responses in mice, causing a significant increase in CD8 + T cells and CD19 + B cells as well as a significant increase in the serum level of specific IgG. The neutralizing antibodies produced in the immunized mice could significantly inhibit the replication of RSV Long strain in vivo. Conclusions:The nanovaccine OMV-preF can induce high-level humoral and cellular immune responses, and the antibodies produced following immunization can effectively inhibit viral replication. This study provides a new strategy for RSV subunit vaccines.

Objective The pathophysiological process of acute high-altitude hypoxia-induced lung injury affects protein expression levels,which are mainly evaluated by Western blot.No systematic study has investigated changes in internal control proteins as calibration loading amounts.Methods Lung injury at an altitude of 6000 m was induced in a low-pressure,low-oxygen chamber for 8,24,and 72 h using C57BL/6J mice.Establishment of the model was confirmed by hematoxylin and eosin staining.Expression levels of various internal control proteins,including vinculin,α-tubulin,eukaryotic translation initiation factor 5(EIF5),β-actin,and glyceraldehyde 3-phosphate dehydrogenase(GAPDH)were detected by Western blot,and total protein expression was detected by Coomassie blue staining.Furthermore,the lung injury model in vitro was establised by using,Bronchial epithelial cell(BZAS-2B)andhunman umbilical vein endothelial cells(HUVECS)confirmed by TUNEL staining.Expression levels of internal control proteins were detected by Western blot,and total protein expression was detected by Coomassie Blue staining.Results Acute 8,24,and 72 h hypoxic models were successfully established in lung tissue,demonstrating consistent total protein expression and stable levels of the internal reference proteins vinculin,α-tubulin,EIF5,andβ-actin.GAPDH expression was elevated in the HH8 h,HH24 h,and HH72 h groups compared with the normoxia(Nor)group,but only the increase at HH72 h groups was significant.Similarly,8,24,and 48 h hypoxic models were successfully established in BEAS-2B cells and HUVECs,with consistent total protein expression.In BEAS-2B cells,expression levels of the internal reference proteins β-actin and GAPDH were consistent with the normoxic control(NC)group,while vinculin,α-tubulin,and EIF5 expression levels were significantly reduced under hypoxic conditions for up to 24 h.In HUVECs,vinculin and α-tubulin expression levels were also consistent with the NC group,while EIF5,β-actin,and GAPDH expression levels were significantly reduced at 8 h and increased at 48 h.Conclusions Acute hypoxia induces lung tissue injury,and protein expression levels of the internal reference proteins vinculin,α-tubulin,EIF5,and β-actin are stable,making them suitable internal references for Western blot.Additionally,Western blot detected differential expression levels of the internal reference proteins vinculin,α-tubulin,EIF5,β-actin,and GAPDH in BEAS-2B cells and HUVECs,as the most important in vitro lung tissue models of hypoxia-induced injury.

Objective The pathophysiological process of acute high-altitude hypoxia-induced lung injury affects protein expression levels,which are mainly evaluated by Western blot.No systematic study has investigated changes in internal control proteins as calibration loading amounts.Methods Lung injury at an altitude of 6000 m was induced in a low-pressure,low-oxygen chamber for 8,24,and 72 h using C57BL/6J mice.Establishment of the model was confirmed by hematoxylin and eosin staining.Expression levels of various internal control proteins,including vinculin,α-tubulin,eukaryotic translation initiation factor 5(EIF5),β-actin,and glyceraldehyde 3-phosphate dehydrogenase(GAPDH)were detected by Western blot,and total protein expression was detected by Coomassie blue staining.Furthermore,the lung injury model in vitro was establised by using,Bronchial epithelial cell(BZAS-2B)andhunman umbilical vein endothelial cells(HUVECS)confirmed by TUNEL staining.Expression levels of internal control proteins were detected by Western blot,and total protein expression was detected by Coomassie Blue staining.Results Acute 8,24,and 72 h hypoxic models were successfully established in lung tissue,demonstrating consistent total protein expression and stable levels of the internal reference proteins vinculin,α-tubulin,EIF5,andβ-actin.GAPDH expression was elevated in the HH8 h,HH24 h,and HH72 h groups compared with the normoxia(Nor)group,but only the increase at HH72 h groups was significant.Similarly,8,24,and 48 h hypoxic models were successfully established in BEAS-2B cells and HUVECs,with consistent total protein expression.In BEAS-2B cells,expression levels of the internal reference proteins β-actin and GAPDH were consistent with the normoxic control(NC)group,while vinculin,α-tubulin,and EIF5 expression levels were significantly reduced under hypoxic conditions for up to 24 h.In HUVECs,vinculin and α-tubulin expression levels were also consistent with the NC group,while EIF5,β-actin,and GAPDH expression levels were significantly reduced at 8 h and increased at 48 h.Conclusions Acute hypoxia induces lung tissue injury,and protein expression levels of the internal reference proteins vinculin,α-tubulin,EIF5,and β-actin are stable,making them suitable internal references for Western blot.Additionally,Western blot detected differential expression levels of the internal reference proteins vinculin,α-tubulin,EIF5,β-actin,and GAPDH in BEAS-2B cells and HUVECs,as the most important in vitro lung tissue models of hypoxia-induced injury.

Objective:To analyze the protective effect of a respiratory syncytial virus (RSV) vaccine based on bacterial outer membrane vesicle (OMV) in mice.Methods:The pre-fusion protein (preF) of RSV was linked to the surface of OMV through the transmembrane protein cytolysin A (ClyA) to form the nanovaccine OMV-preF. The morphological characteristics of OMV and OMV-preF were observed under a transmission electron microscope. OMV-preF was intramuscularly injected into BALB/c mice and the elicited humoral and cellular immune responses were evaluated. The protective effect of OMV-preF was assessed by challenging the immunized mice with RSV Long strain. One-way analysis of variance and Tukey test were used for statistical analysis.Results:The results showed that preF was stably expressed in OMV, and both OMV-preF and OMV exhibited a double-layer vesicle structures under the microscope. OMV-preF could significantly activate the cellular and humoral immune responses in mice, causing a significant increase in CD8 + T cells and CD19 + B cells as well as a significant increase in the serum level of specific IgG. The neutralizing antibodies produced in the immunized mice could significantly inhibit the replication of RSV Long strain in vivo. Conclusions:The nanovaccine OMV-preF can induce high-level humoral and cellular immune responses, and the antibodies produced following immunization can effectively inhibit viral replication. This study provides a new strategy for RSV subunit vaccines.

Objective:To analyze antiviral activity against respiratory syncytial virus (RSV) of interferon (IFN)-α2b and IFN-λ1 on Hep2 cells and human airway epithelial (HAE) cells.Methods:IFN-α2b or IFN-λ1 was incubated with Hep2 cells after RSV infection, and 48 hours later, the cytopathic effect was observed, the viral load was determined using real time/reverse transcription quantitative polymerase chain reaction (RT qPCR), RSV F protein expression was detected using immunofluorescence, and cell survival rate was detected using crystal violet. HAE cells were incubated with IFN-α2b or IFN-λ1 for 24 hours, and then HAE were challenged with RSV. The viral load in the culture supernatant was determined on days 1-7 using RT qPCR, RSV F protein was determined with immunofluorescence and the viral titers in the culture supernatant was detected on day 7 by plaque assay.Results:In Hep2 cells, the CPE of the treatment groups (IFN-α2b and IFN-λ1) was alleviated compared to the virus control group, and the CPE of the high concentration group was lighter than that of the low concentration group. Different concentrations of IFN-α2b and IFN-λ1 could significantly reduce the viral load of RSV ( P<0.001), and the viral load of the high concentration group was significantly lower than that of the low concentration group ( P<0.001). In addition, IFN-α2b and IFN-λ1 could reduce the RSV F protein expression after RSV infection and improve cell survival rate. In HAE cells, IFN-α2b and IFN-λ1 could inhibit RSV virus replication, reduce virus titers ( P<0.001) and reduce RSV F protein expression. Conclusions:IFN-α2b and IFN-λ1 both showed great antiviral activity against RSV in Hep2 and HAE cells, providing data reference for the study of interferon against respiratory viruses.

Objective:To isolate and culture WU polyomavirus (WUPyV), and to analyze the genome-wide evolutionary patterns, homology and population dynamics.Methods:Real-time quantitative PCR was used to detect the nasopharyngeal aspirate samples of hospitalized children with respiratory tract infection in Beijing Friendship Hospital during 2020 to 2022. Primary human airway epithelial cells cultured at the air-liquid interface were used to isolate and culture WUPyV. Whole genome sequence of the isolated strain was obtained by Sanger sequencing. For phylogenetic and evolutionary dynamics analysis, the whole genome was compared with the published whole genome sequences in GenBank database.Results:The detection rate of WUPyV was 4.7% (31/659) during 2020 to 2022, and a clinical strain BJ0593 of WUPyV type Ⅲc was successfully isolated. The homology of the whole genome and gene fragments of WUPyV was high. The average evolutionary rate of VP2 gene was about 1.256×10 -4 substitution/site every year, and the population dynamics of WUPyV tended to be flat in the last decade. Conclusions:This study successfully isolated a clinical WUPyV type Ⅲ strain for the first time, which provided the basis for further investigation on the molecular evolution and pathogenicity of WUPyV.

Objective:To investigate the epidemiological characteristics of human bocavirus 1 (HBoV1) and to analyze the genetic variation.Methods:A total of 2 848 nasopharyngeal aspirate (NPAs) specimens were collected from hospitalized children with acute respiratory tract infections (ARTI) in Beijing Friendship Hospital from April 2017 to March 2019, and HBoV1 was detected by quantitative real-time PCR. Epidemiological analysis was carried out based on the clinical information of the patients. The nested PCR method was used to amplify the NP1 and VP1 genes of HBoV1 for homology analysis. Maximum clade credibility tree (MCC tree) and genetic polymorphism map were constructed to analyze the time evolution of HBoV1 VP1.Results:HBoV1 was detected in 90(3.16%) of 2 848 NPAs, most (93.33%, 84/90) HBoV1-positive cases were among children <5 years of age. HBoV1 could be detected throughout the year with a higher prevalence 7.23% (18/249) in October. Of the 90 HBoV1-infected cases, the main clinical symptoms were fever and cough, 44(48.89%) were co-infected with other respiratory viruses; 55 NP1 sequences and 47 VP1 sequences were obtained by nested PCR amplification, phylogenetic analysis showed that the nucleotide homology was 98.9%~-100% and 99.1%~-100%, respectively. MCC tree showed that the HBoV1 VP1 gene sequence obtained in this study appeared in two adjacent clades, the gene evolution was stable.Conclusions:HBoV1 is one of the common viruses that cause respiratory infection among children in Beijing. HBoV1 genetic evolution is relatively stable, but it still needs to be monitored continuously.

Objective:To construct 2019 novel coronavirus (2019-nCoV) 614D and 614G pseudovirus by HIV lentivirus packaging system and explore their biological specificity.Methods:The recombinant expression plasmids pCDNA3.1-614D and pCDNA3.1-614G were transiently cotransfected with psPAX2 and pLenti CMV Puro LUC into 293T cells respectively. After 72 hours, the supernatant was collected and ultracentrifuged with 20% sucrose cushion. The titer, morphology, protein expression and neutralizing activity of pseudovirus were determined.Results:S protein specific fluorescence was detected by indirect immunofluorescence test, Western blot analysis showed S protein was expressed, and the spike of pseudovirus was observed under transmission electron microscope. The titers of pseudovirus 614D and 614G were 1.12×10 4 and 2.52×10 4 TCID 50/ml, respectively. The pseudovirus 614D and 614G could be neutralized by S rabbit polyclonal antibody, indicating that the pseudovirus has high specificity. Conclusions:In this study, 2019-nCoV 614D and 614G pseudovirus was successfully constructed, which laid the foundation for the establishment of in vitro neutralizing antibody detection platform based on pseudovirus.

Objective:To study the sequence characteristics and genetic variation of a human respiratory syncytial virus (HRSV) subtype A genome in Beijing.Methods:The genomic RNA of HRSV from nasopharyngeal aspirate samples was sequenced and obtained a whole genome sequence of HRSV A subtype. The phylogenetic tree was constructed with reference sequences of other HRSV strains. The major proteins were compared and single nucleotide polymorphism analyzed. In addition, the N-glycosylationsites of F and G protein were predicted.Results:Phylogenetic tree and homology analysis results suggest that the HRSV strain (RSVA/Beijing-China/2017) was the A subtype ON1 genotype. Nucleotide and amino acid variation analysis showed that G protein, F protein and L protein had some substitutions. Analysis of amino acid variation sites showed that amino acid substitution (L142S) occurred at position 142 of G protein. For F protein, there were two substitutions, which were S105N in the P27 peptide (110-136aa) and C69Y in the antigen sites ? (62-69 aa and 196-210 aa). The prediction of N-glycosylation sites revealed that there were 5 N-glycosylation sites of F protein and 4 N-glycosylation sites of G protein in this strain.Conclusions:The HRSV strain obtained in Beijing belongs to A subtype ON1 genotype. The G, F and L proteins have large variations, and 22 amino acid substitutions have occurred in the G and F proteins.

Glucagon-like poptide-1 (GLP-1) is an incretin secreted by the enteroendocrine L cells of the gut. Upon the activation of GLP-1 receptor, adenylyl cyclase is activated and cAMP is generated, leading to the activation of protein kinase A and Epac signal pathway. GLP-1 could also activate calcium/calmodulin pathway as well as mitogen-activated protein kinases and phosphatidyl inositol-3 kinase pathway. GLP-1 not only stimulates the phase 1 and phase 2 insulin secretion, but also increases insulin synthesis. GLP-1 also stimulates proliferation and differentiation of islet β-cells, and protects β-cell from apoptosis and modulates endoplasmic reticulum stress response leading to promotion of β-cell adaptation and survival.