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 explore the clinical phenotype and genetic characteristics of a child with Progressive familial intrahepatic cholestasis type 8 (PFIC8). METHODS A child with PFIC diagnosed at Beijing Children's Hospital Affiliated to Capital Medical University in September 2025 was selected as the study subject. Peripheral venous blood samples were collected from the child and her parents. Following extraction of genomic DNA, whole-exome sequencing (WES) was carried out. Candidate variants were validated by Sanger sequencing. The pathogenicity of the candidate variants was classified based on the guidelines from American College of Medical Genetics and Genomics (ACMG). This study was approved by the Medical Ethics Committee of Beijing Children's Hospital Affiliated to Capital Medical University (Ethics No.: 2023-E-126-Y). RESULTS: The proband, a 2-month-old female infant, had manifested jaundice of the skin and sclera, and slightly distended abdomen. She had no visible abdominal wall varicose veins, soft abdomen, and no palpable masses. Biliary atresia was ruled out by intraoperative cholangiography. WES revealed that she has harbored compound heterozygous variants of KIF12 gene, namely c.809C>T (p.Ala270Val) and c.1313G>A (p.Arg438Lys), which were verified by Sanger sequencing to have derived from her mother and father, respectively. According to the ACMG guidelines, both variants were classified as variants of uncertain significance (VUS). Based on the pre-defined search strategy, 10 articles were retrieved, which involved 25 PFIC cases, including 5 from China. Together with the proband of this study, the 26 PFIC patients have primarily presented with high GGT cholestasis, with the genetic cause in all cases attributed to variants of the KIF12 gene. CONCLUSION The c.809C>T and c.1313G>A compound heterozygous variants of the KIF12 gene probably underlay the pathogenesis of cholestatic liver disease in this child. Above findings have enriched the mutational and phenotypic spectra of PFIC8.
Humans ; Kinesins/genetics* ; Female ; Cholestasis, Intrahepatic/genetics* ; Infant ; Heterozygote ; Mutation ; Exome Sequencing ; Male

Biliary atresia (BA) is a serious neonatal disease, which can eventually develop into cholestatic cirrhosis due to inflammation and progressive fibrosis of bile ducts inside and outside the liver.Kasai portoenterostomy (KP) is the main management strategy of BA that can help children rebuild bile drainage channels.However, the progress of intrahepatic diseases will cause portal hypertension, liver fibrosis, end-stage liver disease and other complications to varying degrees after KP in children, affecting their short-term and long-term prognosis and health.It is also possible that children need liver transplantation within one year after operation.At present, there is no consensus strategy for the prediction of the prognosis of KP in clinical practice.There is a need to find a reliable non-invasive method with high sensitivity and specificity to predict the prognosis after KP.Recently, many examination strategies have been proposed for predicting the prognosis after KP.In this paper, these non-invasive or minimally invasive methods were reviewed according to the source of predictors.

Biliary atresia (BA) is a serious neonatal disease, which can eventually develop into cholestatic cirrhosis due to inflammation and progressive fibrosis of bile ducts inside and outside the liver.Kasai portoenterostomy (KP) is the main management strategy of BA that can help children rebuild bile drainage channels.However, the progress of intrahepatic diseases will cause portal hypertension, liver fibrosis, end-stage liver disease and other complications to varying degrees after KP in children, affecting their short-term and long-term prognosis and health.It is also possible that children need liver transplantation within one year after operation.At present, there is no consensus strategy for the prediction of the prognosis of KP in clinical practice.There is a need to find a reliable non-invasive method with high sensitivity and specificity to predict the prognosis after KP.Recently, many examination strategies have been proposed for predicting the prognosis after KP.In this paper, these non-invasive or minimally invasive methods were reviewed according to the source of predictors.

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.

Influenza B virus (IBV) is more likely to cause complications than influenza A virus (IAV) and even causes higher disease burden than IAV in a certain season, but IBV has received less attention. In order to analyze the genetic evolution characteristics of the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018), we constructed genetic evolution trees and analyzed the homology and different amino acids of hemagglutinin and neuraminidase referring to the vaccine strains recommended by World Health Organization (WHO). We found that strain B/Guangxi-Jiangzhou/1352/2018 was free of interlineage reassortment and poorly matched with the vaccine strain B/Colorado/06/2017 of the same year. We also determined the median lethal dose (LD₅₀) and the pathogenicity of strain B/Guangxi-Jiangzhou/1352/2018 in mice. The results showed that the LD₅₀ was 10^5.9 TCID₅₀ (median tissue culture infective dose), the IBV titer in the lungs reached peak 1 d post infection and the mRNA level of the most of inflammatory cytokines in the lungs reached peak 12 h post infection. The alveoli in the lungs were severely damaged and a large number of inflammatory cells were infiltrated post infection. The study demonstrated that the clinical strain IBV (B/Guangxi-Jiangzhou/1352/2018) could infect mice and induce typical lung inflammation. This will facilitate the research on the pathogenesis and transmission mechanism of IBV, and provide an ideal animal model for evaluation of new vaccines, antiviral and anti-inflammatory drug.
Amino Acids/genetics* ; Animals ; Antiviral Agents/pharmacology* ; China ; Cytokines/metabolism* ; Hemagglutinins/metabolism* ; Humans ; Influenza B virus/pathogenicity* ; Influenza, Human/virology* ; Mice ; Neuraminidase/genetics* ; Orthomyxoviridae Infections/virology* ; Phylogeny ; RNA, Messenger/metabolism* ; Virulence/genetics*

Nonpharmaceutical interventions (NPIs) have been commonly deployed to prevent and control the spread of the coronavirus disease 2019 (COVID-19), resulting in a worldwide decline in influenza prevalence. However, the influenza risk in China warrants cautious assessment. We conducted a cross-sectional, seroepidemiological study in Shandong Province, Northern China in mid-2021. Hemagglutination inhibition was performed to test antibodies against four influenza vaccine strains. A combination of descriptive and meta-analyses was adopted to compare the seroprevalence of influenza antibodies before and during the COVID-19 pandemic. The overall seroprevalence values against A/H1N1pdm09, A/H3N2, B/Victoria, and B/Yamagata were 17.8% (95% CI 16.2%-19.5%), 23.5% (95% CI 21.7%-25.4%), 7.6% (95% CI 6.6%-8.7%), and 15.0 (95% CI 13.5%-16.5%), respectively, in the study period. The overall vaccination rate was extremely low (2.6%). Our results revealed that antibody titers in vaccinated participants were significantly higher than those in unvaccinated individuals (P < 0.001). Notably, the meta-analysis showed that antibodies against A/H1N1pdm09 and A/H3N2 were significantly low in adults after the COVID-19 pandemic (P < 0.01). Increasing vaccination rates and maintaining NPIs are recommended to prevent an elevated influenza risk in China.

Objective: To isolate the cross-reactive antibodies against hemagglutinin of influenza virus and identify its biological function. Methods: The antibodies gene reservoir of cross-reactive and H5N1 pseudotype particles neutralizing B cell circulating in peripheral blood of a human H5N1 case was recovered by in vitro B cell culture, screening, RT-PCR and expression vector cloning techniques. The Ab gene pairing was screened by transient transfection of human kidney 293T cells and detected using ELISA and neutralization test. The heterosubtypic antibodies were prepared and characterized. Results: We discovered the VH1-2-based heterosubtypic antibodies from two B cell lineages could neutralize GX-H5N1 pseudotype particles and have broader binding with Group 1 (including H1, H5, H6 and H9) and H7 subtype. Conclusions Cross-reactive antibodies can be induced by H5N1 infection.

Objective: To develop the monoclonal antibody (mAb) against neuraminidase of H7N9 subtype influenza A virus and identify its biological function. Methods: Female 8 week-old BALB/c mice were immunized and the splenocytes of the mice were fused with Sp2/0 myeloma cells. Indirect ELISA was used to screen hybridoma and the positive clones were subject to be subcloned. Positive clones were identified and the monoclonal antibodies(mAbs) were obtained by purifying the ascetic fluid of mice injected with the hybridoma. The NA-binding as well as neuraminidase-inhibition activity of these mAbs were determined. Results: Three mAbs against neuraminidase of H7N9 subtype influenza A virus, 1G8, 3C4 and 4E8, were obtained. They demonstrated different epitop-recognizing. 3C4 and 4E8 exhibited neuraminidase inhibitory activity, with a IC₅₀ of 1.45 μg/ml and 8.65 μg/ml, respectively. Conclusions The results suggested that mAbs specific to neuraminidase of H7N9 subtype influenza A virus were developed, providing an useful tool in control and preventing the novel H7N9 influenza A virus.