Objective:To analyze the changes in the phylogenetic and antigenic characteristics of the hemagglutinin (HA) gene of influenza virus A(H3N2) [A(H3N2)] during the 2022-2024 influenza seasons in Beijing.Methods:The data of influenza-like cases and A(H3N2) strains from 17 network laboratories and their corresponding sentinel hospitals were collected during the 2022-2024 influenza seasons. The HA genes were amplified and sequenced after extracting nucleic acids of the chosen virus strains. BioEdit, the nucleotide and amino acid sequence identity were conducted, and the maximum likelihood method in MEGA 5.0 software was used to construct the phylogenetic tree of HA genes. Web Logo displayed the amino acid mutation, and the N-glycosylation sites of HA online were analyzed using the NetNGlyc1.0 Server online. The Datamonkey platform was utilized to analyze the positive selection pressure sites of the HA protein.Results:The 2022-2024 influenza season includes 2022-2023 and 2023-2024. During the influenza seasons of 2022-2024, the positive rates of A(H3N2) nucleic acid were 10.35% (2 127/20 543) and 10.47% (4 386/41 876), respectively. In the 2022-2023 influenza season, there were two peaks in the A(H3N2). The comparison of HA genes between all A(H3N2) strains studied with the 2022-2024 vaccine strain (A/Darwin/9/2021) revealed that all of the strains studied have the two amino acid mutations involving 186 and 225 receptor binding sites. There were 31 amino acid substitutions in the 2022-2023 influenza season, of which 18 variant sites involved antigenic determinants. There were 35 amino acid mutations during the 2023-2024 influenza season, of which 14 were related to antigenic determinants. There were changes in the genetic evolutionary subclades of A(H3N2) strains in two influenza seasons: from 2022 to 2023, three evolutionary subclades were co-prevalent together, with the 3C.2a1b.2a.2a.3a.1 accounting for 76.67% (23/30), the 3C.2a1b.2a.1a accounting for 20.00% (6/30), the 3C.2a1b.2a.2a.1 accounting for 3.33% (1/30); from 2023 to 2024, two subclades were prevalent, with 3C.2a1b.2a.2a.3a.1 accounting for 95.12% (39/41) and 3C.2a1b.2a.2a.1 accounting for 4.88% (2/41). The glycosylation site changes of the HA protein of A(H3N2) have been enhanced from 2023 to 2024. The 145 amino acid position of the HA protein of the A(H3N2) was the positive selection site for stress selection site analysis.Conclusions:The evolutionary subclades of the HA gene of A(H3N2) in Beijing showed changes from 2022 to 2024, and the glycosylation site polymorphism of the HA protein of A(H3N2) significantly increased from 2023 to 2024. Continuous monitoring of HA mutations in the A(H3N2) is crucial, providing a basis for developing influenza prevention and control strategies, as well as new strategic support for screening influenza vaccine components, vaccine design, and discovery of drug targets.

ObjectiveTo establish an adjusted Serfling regression model to estimate the excess cases and the excess epidemic period of hand-foot-mouth disease (HFMD) in Beijing from 2011 to 2019, so as to provide data support and decision-making basis for HFMD prevention and control. MethodsThe weekly number of HFMD cases in Beijing from 2011 to 2019 was utilized for adjusted the Serfling regression model. Then the adjusted model was used to fit the baseline and epidemic threshold of HFMD in Beijing from 2011 to 2019, calculating the excess cases and determining the excess epidemic period. ResultsA total of 279 306 cases of HFMD were reported in Beijing from 2011 to 2019, with the climax of the disease occurring in summer and autumn. After adjusting the fitting R² of the Serfling regression model to 0.773, a total of 10 excess epidemic periods totaling 92 weeks were estimated, mainly occurring in summer. The highest number of excess cases during an excess epidemic period was found in 2014 (1 272 cases, 95%CI: 990‒1 554), accounting for 65.04% of the actual cases (95%CI: 50.62%‒79.46%). ConclusionThe adjusted Serfling regression model fits well and can be utilized for early warning of HFMD and estimating the disease burden caused by HFMD.

OBJECTIVE: To investigate the role of α7-nicotinic acetylcholine receptor (α7-nAChR) in the regulation of neuronal activity and expression of synapse-related proteins in the thalamic reticular nucleus (TRN) of insomnia rats treated by acupuncture. METHODS: A total of 36 male Sprague-Dawley (SD) rats of clean grade were randomly divided into a control group, a model group, an acupuncture group, and an acupuncture+antagonist group, with 9 rats in each group. The model group, the acupuncture group, and the acupuncture+antagonist group were treated with intraperitoneal injection of p-chlorophenylalanine (PCPA) to establish insomnia model. After successful modeling, the acupuncture group and the acupuncture+antagonist group received acupuncture at bilateral Neiguan (PC6) and Zusanli (ST36) once daily for 5 consecutive days. Thirty min before each acupuncture session, the acupuncture+antagonist group was intraperitoneally injected with methyllycaconitine citrate (MLA), an α7-nAChR antagonist, at a dosage of 5 mg/kg while the acupuncture group received the same volume of 0.9% sodium chloride solution. The rats' daytime spontaneous activity was observed. Neuronal discharge in the TRN was detected using neuroelectrophysiological methods. Immunofluorescence staining was used to detect parvalbumin-positive (PV⁺) neurons and co-expression of PV⁺ and postsynaptic density protein-95 (PSD-95) in the TRN. RESULTS: Compared with the control group, the model group showed increased daytime spontaneous activity (P<0.01); decreased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.01), prolonged inter-discharge intervals (P<0.01) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). Compared with the model group, the acupuncture group showed decreased daytime spontaneous activity (P<0.01); increased average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); increased neuronal discharge frequency (P<0.01), shortened inter-discharge intervals (P<0.01) in the TRN; increased number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.05). Compared with the acupuncture group, the acupuncture+antagonist group exhibited increased daytime spontaneous activity (P<0.01); reduced average fluorescence intensity and positive number of PV⁺ neurons in the TRN (P<0.01); decreased neuronal discharge frequency (P<0.05), prolonged inter-discharge intervals (P<0.05) in the TRN; reduced number of PV⁺/PSD-95 double-positive cells in the TRN (P<0.01). CONCLUSION α7-nAChR are involved in mediating the regulatory effect of acupuncture on circadian rhythm disturbances in PCPA-induced insomnia rats. Blocking α7-nAChR attenuates the activating effect of acupuncture on TRN neurons, and reduces the expression of PSD-95 protein on GABAergic neurons.
Animals ; Male ; Acupuncture Therapy ; alpha7 Nicotinic Acetylcholine Receptor/genetics* ; Rats, Sprague-Dawley ; Rats ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Neurons/metabolism* ; Humans ; Thalamic Nuclei/physiopathology* ; Acupuncture Points ; Disks Large Homolog 4 Protein

Objective:To analyze the changes in the phylogenetic and antigenic characteristics of the hemagglutinin (HA) gene of influenza virus A(H3N2) [A(H3N2)] during the 2022-2024 influenza seasons in Beijing.Methods:The data of influenza-like cases and A(H3N2) strains from 17 network laboratories and their corresponding sentinel hospitals were collected during the 2022-2024 influenza seasons. The HA genes were amplified and sequenced after extracting nucleic acids of the chosen virus strains. BioEdit, the nucleotide and amino acid sequence identity were conducted, and the maximum likelihood method in MEGA 5.0 software was used to construct the phylogenetic tree of HA genes. Web Logo displayed the amino acid mutation, and the N-glycosylation sites of HA online were analyzed using the NetNGlyc1.0 Server online. The Datamonkey platform was utilized to analyze the positive selection pressure sites of the HA protein.Results:The 2022-2024 influenza season includes 2022-2023 and 2023-2024. During the influenza seasons of 2022-2024, the positive rates of A(H3N2) nucleic acid were 10.35% (2 127/20 543) and 10.47% (4 386/41 876), respectively. In the 2022-2023 influenza season, there were two peaks in the A(H3N2). The comparison of HA genes between all A(H3N2) strains studied with the 2022-2024 vaccine strain (A/Darwin/9/2021) revealed that all of the strains studied have the two amino acid mutations involving 186 and 225 receptor binding sites. There were 31 amino acid substitutions in the 2022-2023 influenza season, of which 18 variant sites involved antigenic determinants. There were 35 amino acid mutations during the 2023-2024 influenza season, of which 14 were related to antigenic determinants. There were changes in the genetic evolutionary subclades of A(H3N2) strains in two influenza seasons: from 2022 to 2023, three evolutionary subclades were co-prevalent together, with the 3C.2a1b.2a.2a.3a.1 accounting for 76.67% (23/30), the 3C.2a1b.2a.1a accounting for 20.00% (6/30), the 3C.2a1b.2a.2a.1 accounting for 3.33% (1/30); from 2023 to 2024, two subclades were prevalent, with 3C.2a1b.2a.2a.3a.1 accounting for 95.12% (39/41) and 3C.2a1b.2a.2a.1 accounting for 4.88% (2/41). The glycosylation site changes of the HA protein of A(H3N2) have been enhanced from 2023 to 2024. The 145 amino acid position of the HA protein of the A(H3N2) was the positive selection site for stress selection site analysis.Conclusions:The evolutionary subclades of the HA gene of A(H3N2) in Beijing showed changes from 2022 to 2024, and the glycosylation site polymorphism of the HA protein of A(H3N2) significantly increased from 2023 to 2024. Continuous monitoring of HA mutations in the A(H3N2) is crucial, providing a basis for developing influenza prevention and control strategies, as well as new strategic support for screening influenza vaccine components, vaccine design, and discovery of drug targets.

Objective:To compare the enrichment effects of ultrafiltration, polyethylene glycol (PEG) precipitation, aluminum salt precipitation, and anionic membrane adsorption-elution on viruses in drinking water.Methods:Using phage MS2 as the target virus, three different concentrations of drinking water samples were prepared, and the samples were enriched by ultrafiltration 1, ultrafiltration 2, PEG precipitation, aluminum salt precipitation, and anionic membrane adsorption-elution method, respectively. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to quantify MS2 nucleic acid in pre and post concentrated samples and the recovery rates of MS2 in samples with high, medium and low concentrations were compared among the five methods.Results:Comparing the MS2 enrichment recovery rates of individual enrichment method in water samples of different concentrations, ultrafiltration method 1, PEG precipitation method, aluminum salt precipitation method, and membrane adsorption-elution method were not affected by the sample concentration, and the differences of the recovery rates for the three concentration water samples among the four methods were not statistically significant ( P>0.05). The MS2 enrichment recovery rates of the five enrichment methods were significantly different in all concentration samples ( P<0.05). The recovery rates of ultrafiltration method 1 were higher in all three concentration samples, followed by aluminum salt precipitation and anionic membrane adsorption-elution, PEG precipitation were higher in high concentration samples, but lower in low and medium concentration samples, and the recovery rates of ultrafiltration method 2 were the lowest in all three concentration samples. Comparing the Ct values of MS2 in the enriched samples by five methods, the Ct values of ultrafiltration method 1 were the smallest in the three concentration water samples. There was no statistically significant difference in MS2 Ct values among the five enrichment methods in the medium and high concentration water samples ( P>0.05). In low concentration simulated water samples, only the difference of MS2 Ct value between ultrafiltration method 1 and ultrafiltration method 2 was statistically significant ( Z=16.000, P=0.016). Conclusions:Considering the operation simplicity, operation time and virus recovery rate after enrichment, ultrafiltration was the most effective method for virus enrichment in drinking water.

Objective:To evaluate the preservation efficacy of 7 non-inactivating virus preservation solutions.Methods:Equal amounts of H1N1 virus were added to 7 commercially available non-inactivating virus preservation solutions, and the samples were stored at -20 ℃, 4 ℃, 25 ℃ and 37 ℃ for 1 hour, 6 hours, 1 day, 3 days, and 5 days. The viral nucleic acid in each simulated sample under different storage conditions was measured using real-time quantitative PCR. The hemagglutination (HA) titer was determined through viral isolation culture and hemagglutination assay, comparing the differences in viral growth activity across different storage solutions and conditions.Results:Except for solution E, the other solutions effectively protected viral nucleic acid at the 4 storage temperatures. In terms of viral activity, solutions A, B, C, and D effectively maintained viral viability. A and B showing the best performance, E and F showed poorer performance, and G performed the worst.Conclusions:Most non-inactivating virus preservation solutions effectively protect viral nucleic acid, but there are significant differences in their ability to maintain viral viability. To ensure optimal virus preservation, it is recommended that medical institutions evaluate the effectiveness of preservation solutions before use.

Objective:To compare the enrichment effects of ultrafiltration, polyethylene glycol (PEG) precipitation, aluminum salt precipitation, and anionic membrane adsorption-elution on viruses in drinking water.Methods:Using phage MS2 as the target virus, three different concentrations of drinking water samples were prepared, and the samples were enriched by ultrafiltration 1, ultrafiltration 2, PEG precipitation, aluminum salt precipitation, and anionic membrane adsorption-elution method, respectively. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to quantify MS2 nucleic acid in pre and post concentrated samples and the recovery rates of MS2 in samples with high, medium and low concentrations were compared among the five methods.Results:Comparing the MS2 enrichment recovery rates of individual enrichment method in water samples of different concentrations, ultrafiltration method 1, PEG precipitation method, aluminum salt precipitation method, and membrane adsorption-elution method were not affected by the sample concentration, and the differences of the recovery rates for the three concentration water samples among the four methods were not statistically significant ( P>0.05). The MS2 enrichment recovery rates of the five enrichment methods were significantly different in all concentration samples ( P<0.05). The recovery rates of ultrafiltration method 1 were higher in all three concentration samples, followed by aluminum salt precipitation and anionic membrane adsorption-elution, PEG precipitation were higher in high concentration samples, but lower in low and medium concentration samples, and the recovery rates of ultrafiltration method 2 were the lowest in all three concentration samples. Comparing the Ct values of MS2 in the enriched samples by five methods, the Ct values of ultrafiltration method 1 were the smallest in the three concentration water samples. There was no statistically significant difference in MS2 Ct values among the five enrichment methods in the medium and high concentration water samples ( P>0.05). In low concentration simulated water samples, only the difference of MS2 Ct value between ultrafiltration method 1 and ultrafiltration method 2 was statistically significant ( Z=16.000, P=0.016). Conclusions:Considering the operation simplicity, operation time and virus recovery rate after enrichment, ultrafiltration was the most effective method for virus enrichment in drinking water.

Objective:To evaluate the preservation efficacy of 7 non-inactivating virus preservation solutions.Methods:Equal amounts of H1N1 virus were added to 7 commercially available non-inactivating virus preservation solutions, and the samples were stored at -20 ℃, 4 ℃, 25 ℃ and 37 ℃ for 1 hour, 6 hours, 1 day, 3 days, and 5 days. The viral nucleic acid in each simulated sample under different storage conditions was measured using real-time quantitative PCR. The hemagglutination (HA) titer was determined through viral isolation culture and hemagglutination assay, comparing the differences in viral growth activity across different storage solutions and conditions.Results:Except for solution E, the other solutions effectively protected viral nucleic acid at the 4 storage temperatures. In terms of viral activity, solutions A, B, C, and D effectively maintained viral viability. A and B showing the best performance, E and F showed poorer performance, and G performed the worst.Conclusions:Most non-inactivating virus preservation solutions effectively protect viral nucleic acid, but there are significant differences in their ability to maintain viral viability. To ensure optimal virus preservation, it is recommended that medical institutions evaluate the effectiveness of preservation solutions before use.

BACKGROUND:Tissue engineering has brought new hope to the clinical challenge of liver failure,and the preparation of plant-derived decellularized fiber scaffolds holds significant importance in liver tissue engineering. OBJECTIVE:To prepare apple tissue decellularized scaffold material by using fresh apple slices and a solution of sodium dodecyl sulfate,and assess its biocompatibility. METHODS:Fresh apples were subjected to decellularization using phosphate buffer saline and sodium dodecyl sulfate solution,separately.Afterwards,the decellularized apple tissues and apple decellularized scaffold materials were decontaminated with phosphate buffer saline.Subsequently,scanning electron microscopy was used to assess the effectiveness of decellularization of the apple materials.Adipose-derived mesenchymal stem cells were extracted from the inguinal fat BALB/C of mice,and their expression of stem cell-related markers(CD45,CD34,CD73,CD90,and CD105)was identified through flow cytometry.The cells were then divided into a scaffold-free control group and a scaffold group.Equal amounts of adipose-derived mesenchymal stem cells were seeded onto both groups.The biocompatibility of the decellularized scaffold with adipose-derived mesenchymal stem cells was evaluated using CCK-8 assay,hematoxylin-eosin staining,and phalloidine staining.Cell adhesion and growth on the scaffold were observed under light microscopy and scanning electron microscopy.Furthermore,the scaffold was subdivided into the non-induced group and the hepatogenic-induced group.Adipose-derived mesenchymal stem cells were cultured on the decellularized apple scaffold,and they were cultured for 14 days in regular culture medium or hepatogenic induction medium for comparison.Immunofluorescent staining using liver cell markers,including albumin,cytokeratin 18,and CYP1A1,was performed.Enzyme-linked immunosorbent assay was used to detect the secretion of alpha fetoprotein and albumin.Additionally,scanning electron microscopy was employed to observe the morphology of the induced cells on the scaffold,verifying the expression of liver cell-related genes on the decellularized scaffold material.Finally,the cobalt-60 irradiated and sterilized decellularized apple scaffolds were transplanted onto the surface of mouse liver and the degradation of the scaffold was observed by gross observation and hematoxylin-eosin staining after 28 days. RESULTS AND CONCLUSION:(1)The scanning electron microscopy results revealed that the decellularized apple scaffold material retained a porous structure of approximately 100 μm in size,with no residual cells observed.(2)Through flow cytometry analysis,the cultured cells were identified as adipose-derived mesenchymal stem cells.(3)CCK-8 assay results demonstrated that the prepared decellularized apple tissue scaffold material exhibited no cytotoxicity.Hematoxylin-eosin staining and phalloidine staining showed that adipose-derived mesenchymal stem cells were capable of adhering and proliferating on the decellularized apple tissue scaffold.(4)The results obtained from immunofluorescence staining and enzyme-linked immunosorbent assay revealed that adipose-derived mesenchymal stem cells cultured on the decellularized apple scaffolds exhibited elevated expression of liver-specific proteins,including albumin,alpha-fetoprotein,cytokeratin 18,and CYP1A1.These results suggested that they were induced differentiation into hepatocyte-like cells possessing functional characteristics of liver cells.(5)The decellularized apple scaffold implanted at 7 days has integrated with the liver,with partial degradation of the scaffold observed.By 28 days,the decellularized apple scaffold has completely degraded and has been replaced by newly-formed tissue.(6)The results indicate that the decellularized scaffold material derived from apple tissue demonstrates favorable biocompatibility,promoting the proliferation,adhesion,and hepatic differentiation of adipose-derived mesenchymal stem cells.