Reduning is a Chinese patent medicine made from three traditional Chinese herbs： Artemisia caruifolia Buch.-Ham. ex Roxb.， Lonicera japonica Thunb. and Gardenia jasminoides J. Ellis. It has the effect of clearing heat and detoxifying and is widely used in the treatment of respiratory diseases. This article collates the main components of 70 kinds of heat-toxin，including iridoids，lignans，etc. By reviewing the domestic and foreign literature related to influenza and heat-toxin in recent years，this article reviews chemical composition，pharmacological effects，clinical applications and current predicaments of Reduning，and further discusses the differences between the "multi-component-multi-pathway" of heat-toxin and the single-target mechanism of the drug.

Reduning is a Chinese patent medicine made from three traditional Chinese herbs： Artemisia caruifolia Buch.-Ham. ex Roxb.， Lonicera japonica Thunb. and Gardenia jasminoides J. Ellis. It has the effect of clearing heat and detoxifying and is widely used in the treatment of respiratory diseases. This article collates the main components of 70 kinds of heat-toxin，including iridoids，lignans，etc. By reviewing the domestic and foreign literature related to influenza and heat-toxin in recent years，this article reviews chemical composition，pharmacological effects，clinical applications and current predicaments of Reduning，and further discusses the differences between the "multi-component-multi-pathway" of heat-toxin and the single-target mechanism of the drug.

Objective: To analyze respiratory syncytial virus(RSV) outbreaks surveillance results and the epidemiological characteristics in kindergarten and school in Shenzhen during 2017-2023 , so as to provide a scientific reference for control and prevention of RSV. Methods: Epidemiological data and surveillance results of RSV outbreaks in kindergarten and school from 2017 to 2023 were collected for descriptive analyses. Results: A total of 31 RSV outbreaks were identified in kindergarten and school in 2017-2023 in Shenzhen, 346 cases were reported, the average incidence rate was 22.02%. The most annual RSV outbreaks were reported in 2020 with 14 outbreaks, followed by 8 outbreaks in 2023. A total of 64.52% of RSV outbreaks were identified in kindergarten with rest occurring in primary school or middle school. The greatest monthly count of outbreak was 18 (58.06%) in September, followed by 3 outbreaks (9.68%) in March and October. A total of 244 swab samples were collected, 169 samples were positive for respiratory viruses, the positive rate was 69.26%, 121 samples were positive for RSV,from 31 respiratory syncytical virus outbreaks 57 and samples were positive for other respiratory viruses(9 samples were positive for two respiratory viruses). A toral of 14(45.16%) outbreaks are caused by RSV alone, 17 outbreaks (54.84%) were caused by RSV and other respiratory viruses. Conclusions Most RSV outbreaks in kindergarten and school are reported after 2020 in Shenzhen, most RSV outbreaks occur in kindergarten, peak seasons of RSV outbreaks are autumn and spring.

Objective:This study aimed to explore the molecular mechanisms of the maladaptation of H3N2 influenza virus in chicken embryos, provide a theoretical basis for the restoration of H3N2 influenza vaccine production in chicken embryos.Methods:Samples of respiratory secretions from patients with influenza-like symptoms (Influenza-like Illness, ILI) caused by H3N2 influenza virus were inoculated into chicken embryos and Madin-Darby Canine Kidney cells (MDCK), respectively. After isolating the virus, hemagglutination experiments were conducted to detect hemagglutination titers and hemagglutination inhibition experiments were used to compare antigenic differences; further, whole-genome sequencing of H3N2 influenza virus was performed using second-generation high-throughput gene sequencing (Next Generation High-Throughput Gene Sequencing, NGS), and key amino acid sites of mutations were identified through sequence alignment; combined with sialic acid receptor binding experiments, the differences in the binding of wild-type and mutant receptor binding sites (RBS) to sialic acid receptors were compared; finally, molecular docking and molecular dynamics simulation method were used to explore the specific molecular mechanisms of how mutation sites affect the differences in the affinity of the RBS pocket for sialic acid receptors.Results:The hemagglutination assay result indicated that both chicken embryos and MDCK cells could isolate the influenza virus, and the hemagglutination inhibition test showed that no antigenic differences were produced in the isolated strains. NGS analysis revealed that the H3N2 virus underwent an F195Y mutation in the (RBS) region of the hemagglutinin (HA) protein after adaptation through chicken embryo passages. Receptor-binding experiments demonstrated that the F195Y mutation enhanced the virus′s binding ability to α2, 3-linked sialic acid glycan (Neu5Acα2-3Galβ1-4GlcNAcβ-PAA, 3′SLN), while the mutation did not affect the affinity of the RBS pocket for α2, 6-linked sialic acid glycan (Neu5Acα2-6Galβ1-4GlcNAcβ-PAA, 6′SLN). Molecular docking and molecular dynamics simulation result indicate that the F195Y mutation, by replacing a hydrophobic amino acid with a hydrophilic one, leads to a significant decrease in the structure of the RBS pocket, enhancing the binding stability of the H3N2 influenza virus with α2, 3-sln. This is specifically manifested by an increase in binding time and an increase in the number of hydrogen bonds at the RBS site with the receptor. Furthermore, the F195Y mutation does not alter the binding of the virus to other receptors.Conclusions:The F195Y mutation in the RBS pocket of H3N2 influenza virus is a key site affecting the viral chicken embryo inadaptability.

Objective:This study aimed to explore the molecular mechanisms of the maladaptation of H3N2 influenza virus in chicken embryos, provide a theoretical basis for the restoration of H3N2 influenza vaccine production in chicken embryos.Methods:Samples of respiratory secretions from patients with influenza-like symptoms (Influenza-like Illness, ILI) caused by H3N2 influenza virus were inoculated into chicken embryos and Madin-Darby Canine Kidney cells (MDCK), respectively. After isolating the virus, hemagglutination experiments were conducted to detect hemagglutination titers and hemagglutination inhibition experiments were used to compare antigenic differences; further, whole-genome sequencing of H3N2 influenza virus was performed using second-generation high-throughput gene sequencing (Next Generation High-Throughput Gene Sequencing, NGS), and key amino acid sites of mutations were identified through sequence alignment; combined with sialic acid receptor binding experiments, the differences in the binding of wild-type and mutant receptor binding sites (RBS) to sialic acid receptors were compared; finally, molecular docking and molecular dynamics simulation method were used to explore the specific molecular mechanisms of how mutation sites affect the differences in the affinity of the RBS pocket for sialic acid receptors.Results:The hemagglutination assay result indicated that both chicken embryos and MDCK cells could isolate the influenza virus, and the hemagglutination inhibition test showed that no antigenic differences were produced in the isolated strains. NGS analysis revealed that the H3N2 virus underwent an F195Y mutation in the (RBS) region of the hemagglutinin (HA) protein after adaptation through chicken embryo passages. Receptor-binding experiments demonstrated that the F195Y mutation enhanced the virus′s binding ability to α2, 3-linked sialic acid glycan (Neu5Acα2-3Galβ1-4GlcNAcβ-PAA, 3′SLN), while the mutation did not affect the affinity of the RBS pocket for α2, 6-linked sialic acid glycan (Neu5Acα2-6Galβ1-4GlcNAcβ-PAA, 6′SLN). Molecular docking and molecular dynamics simulation result indicate that the F195Y mutation, by replacing a hydrophobic amino acid with a hydrophilic one, leads to a significant decrease in the structure of the RBS pocket, enhancing the binding stability of the H3N2 influenza virus with α2, 3-sln. This is specifically manifested by an increase in binding time and an increase in the number of hydrogen bonds at the RBS site with the receptor. Furthermore, the F195Y mutation does not alter the binding of the virus to other receptors.Conclusions:The F195Y mutation in the RBS pocket of H3N2 influenza virus is a key site affecting the viral chicken embryo inadaptability.

Objective:To understand the epidemiological characteristics of human respiratory syncytial virus (HRSV) among cases presenting with influenza-like illness (ILI) in Shenzhen City from 2019 to 2023.Methods:Respiratory specimens were collected from two national sentinel hospitals in Shenzhen from March 2019 to December 2023, specifically targeting cases of ILI. The real-time PCR method was used for the detection and genotyping of HRSV. Basic demographic information was collected and used for the epidemiological analysis.Results:A total of 9 278 respiratory specimens of influenza-like cases were collected and detected, with a total positive rate of 4.77% (443/9 278) for HRSV. In 2021 (8.48%, 167/1 970), the positive rate of HRSV was significantly higher than in 2019 (3.35%, 52/1 552), 2022 (1.80%, 39/2 169), and 2023 (4.49%, 133/2 960), and the difference was statistically significant ( χ 2=102.395, P<0.001). The prevalence of HRSV was mainly in summer and early autumn (September), and there was an abnormal increase in the positive rate of HRSV in winter 2022. The highest positive rate of HRSV was in children under five years old (9.84%, 330/335). The typing results showed that in 2022, the prevalence of HRSV-A was predominant (71.79%, 28/39), and in 2023, HRSV-A and HRSV-B subtypes coexisted. Conclusions:The prevalence of HRSV in Shenzhen from 2019 to 2023 has obvious seasonality, mainly in summer and early autumn. Children under five years old are the main population of HRSV infections.

Objective: To analyze the monitoring results and characteristics of human metapneumovirus (HMPV) outbreaks in Shenzhen in kindergartens and schools during 2017-2023, so as to provide a evidence for prevention and control of respiratory diseases. Methods: Based on the epidemiological data and laboratory test results of HMPV outbreaks in kindergartens and schools of Shenzhen during 2017 to 2023, collected from the influenza monitoring network platform, Mann Kendall test was applied for trend analysis pf HMPV. Results: A total of 11 HMPV outbreaks were identified in kindergartens and schools from 2017 to 2023 for 128 cases with an average incidence rate of 26.61% in 2018. The most yearly HMPV outbreaks were reported in 2020 being 4 outbreaks, followed by 2 outbreaks in 2018. A total of 72.73% (8 outbreaks) of HMPV outbreaks were identified in kindergartens with 27.27% (3 outbreaks) occurring in primary schools. The monthly greatest count of outbreak was 3(27.27%) in March and December, followed by 2 outbreaks (18.18%) in April and June. A total of 98 swab samples were collected, and 80 samples were positive for respiratory viruses with a positive rate of 81.63%; among them, 67 samples were positive for HMPV alone, 10 samples were positive for other respiratory viruses alone, and 3 samples were positive both for HMPV and other respiratory virus. Among the 11 human metalung virus cases, 6 HMPV outbreaks were caused by human metapneumovirus alone, and 5 outbreaks were caused by HMPV and other respiratory viruses. Except for 2020, the percentage of HMPV outbreaks in influenza like illness outbreaks increased with year ( Z=2.25, P =0.02). Conclusion HMPV outbreaks have been reported every year from 2017 to 2023 in kindergartens and schools in Shenzhen, most of HMPV in kindergartens, and the peak seasons of HMPV outbreaks are spring and winter.

Objective:To understand the epidemiological characteristics of human respiratory syncytial virus (HRSV) among cases presenting with influenza-like illness (ILI) in Shenzhen City from 2019 to 2023.Methods:Respiratory specimens were collected from two national sentinel hospitals in Shenzhen from March 2019 to December 2023, specifically targeting cases of ILI. The real-time PCR method was used for the detection and genotyping of HRSV. Basic demographic information was collected and used for the epidemiological analysis.Results:A total of 9 278 respiratory specimens of influenza-like cases were collected and detected, with a total positive rate of 4.77% (443/9 278) for HRSV. In 2021 (8.48%, 167/1 970), the positive rate of HRSV was significantly higher than in 2019 (3.35%, 52/1 552), 2022 (1.80%, 39/2 169), and 2023 (4.49%, 133/2 960), and the difference was statistically significant ( χ 2=102.395, P<0.001). The prevalence of HRSV was mainly in summer and early autumn (September), and there was an abnormal increase in the positive rate of HRSV in winter 2022. The highest positive rate of HRSV was in children under five years old (9.84%, 330/335). The typing results showed that in 2022, the prevalence of HRSV-A was predominant (71.79%, 28/39), and in 2023, HRSV-A and HRSV-B subtypes coexisted. Conclusions:The prevalence of HRSV in Shenzhen from 2019 to 2023 has obvious seasonality, mainly in summer and early autumn. Children under five years old are the main population of HRSV infections.

Objective:To investigate the role and possible molecular mechanisms of m6A methyltransferase METTL3 and demethylase FTO in H7N9 virus infection.Methods:Western blot(WB) was used to detect the expression of METTL3 and FTO after H7N9 virus infected A549 cells. After knocking down and over-expressing METTL3 and FTO, H7N9 virus infection was tested by WB and TCID 50 for its effect on virus replication. Transcriptome sequencing method were used to analyze differentially expressed genes at the transcriptome level in METTL3 or FTO knockdown and wild-type control cells, and preliminary analysis of potential targets was performed. Results:After 24 h of H7N9 virus infection in A549 cells, METTL3 expression was up-regulated, and FTO did not significantly change. After knocking down METTL3 and FTO, the virus nucleoprotein (NP) expression level and virus titer decreased significantly. Consistent with the result of the knockdown experiments, we found that the viral titer was increased by METTL3 and FTO overexpression. Transcriptome sequencing result showed that 314 and 555 differentially expressed genes were found between the METTL3 or FTO knockdown group and the control group, respectively. GO functional enrichment analysis and KEGG pathway enrichment analysis showed that these genes were related to the host immune response.Conclusions:METTL3 and FTO may play a key role in H7N9 virus infection by regulating host-virus interactions.

Objective: Exploring the molecular characteristic of global and Shenzhen district H5N6 and H7N9 influenza viruses HA untranslated regions(UTRs). Methods: Mega7.0 and DNAStar 7.1.0 were used to construct phylogenetic tree and nucleotide analysis. Results: From 2014 to 2015, 3 strains of H5N6 influenza virus from Shenzhen were compared with the other H5NX influenza viruses, the nucleotide homology of HA-3’UTR was 77.4%-100%, which did not have obvious mutated sites. The nucleotide homology of H5N6-HA-5’UTR was 91.7%-100%, and the sites of 24 and 31 sites were mutated. From 2013 to 2014, 11 strains of H7N9 influenza virus from Shenzhen were compared with the other H7NX influenza viruses, the nucleotide homology of H7N9-HA-5’UTR was 76.8%-100%, which had multi-mutated sites on 2-6, 9, 10, 12 and 15-17 positions. Conclusions HA-UTR from human-infected H5N6 and H7N9 influenza viruses isolated in Shenzhen district has unique molecular characteristics, its conserved region has relatively high homology and the segment-specific region has genetic polymorphism.