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.

Objective:To investigate the effects of pre-existing antibody on seroconversion rate after influenza vaccination.Methods:This study recruited 1 900 healthy volunteers to receive influenza split vaccines in Xinjiang Uygur Autonomous region and Yunnan Province from September 2009 to October 2018. Hemagglutinin agglutination inhibition assay was used to detect the titers of specific antibodies in blood samples collected before vaccination and 28 d after vaccination and the effects of pre-existing antibody on the seroconversion to different influenza vaccine components were analyzed.Results:Trend analysis showed that with the increasing titer of pre-existing antibody, the seroconversion rates to A/H1N1, A/H3N2, B/Victoria and B/Yamagata vaccine components were gradually decreased (χ 2=121.76, P<0.001; χ 2=67.58, P<0.001; χ 2=45.25, P<0.001; χ 2=54.55, P<0.001). After adjusting for factors such as region, gender and age, multivariate logistic regression showed that pre-existing antibody titer equal to or higher than 40 was an independent factor that affected the seroconversion to A/H1N1, A/H3N2 and B/Victoria vaccine components, and the adjusted OR (95%CI) values were 2.50(2.00-3.13)、1.64(1.35-2.00) and 2.50(1.79-3.45), respectively. Conclusions:The seroconversion rate to each vaccine component was negatively correlated with the pre-existing antibody titer. The factor that pre-existing antibody titer equal to or higher than 40 was detrimental to the seroconversion to A/H1N1, A/H3N2 and B/Victoria vaccine components, but had no significant influence on B/Yamagata seroconversion.

Objective:The antigenic and genetic characteristics of influenza A(H1N1)pdm09 virus isolated from the mainland of China during the 2018-2019 influenza surveillance year were analyzed.Methods:Two thousand nine hundred and fifty-eight influenza A(H1N1)pdm09 virus strains in the 2018-2019 influenza surveillance year were analyzed by hemagglutination inhibition test. The hemagglutinin(HA) gene of 279 influenza A(H1N1)pdm09 virus strains was sequenced and analyzed. The representative strains of the dominant clades were performed for antigenic characteristics using post-vaccination human antisera.Results:Two thousand eight hundred and sixty-one (97%, 2 861/2 958) viruses characterized were antigenically similar to A/Michigan/45/2015. All HA gene of the sequenced viruses belonged to 6B.1 clade, and 269(96%, 269/279) viruses belonged to 6B.1A subclade. Compared with the vaccine virus, it had the common amino acid substitutions of S74R, S164T and I295V in the HA protein. There were several small groups with common amino acid substitutions in the 6B.1A subclade, and 51% sequenced viruses had S183P amino acid substitution in this subclade. The result of antigenic analysis using post-vaccination human antiserums showed that most of the representative strains were well inhibited by the sera.Conclusions:The antigenicity of influenza A(H1N1)pdm09 viruses in the mainland of China in 2018-2019 influenza surveillance year matched well with the corresponding vaccine strain, but the HA gene had genetically diverse characteristic.

Objective: Influenza H1N1 subtype vaccine candidate strains from a 2015—2016 year epidemic strain in China were prepared and identified by themethod of classical reassortment. Methods: The influenza H1N1 epidemic strain and H3N2 high-yield reassortant parental strain (X-157) were mixed and inoculated into embryonated chicken eggs by the classical reassortmentmethod . The negative selection of mixed culture virus was carried out with the antiserum of H3 protein and the antiserum of X-157 strain. Real-time PCRmethod was used to test the HA and NA genes. Restriction enzyme digestionmethod was used to identify the internal genes. HA and NA genes of selected strains were sequenced. The strain which HA and NA genes possessed the same amino acid constitution with the wild type virus was selected and immunized to ferret. Two-way test was carried out. Results: Five strains with expected HA and NA genes were selected by real-time PCR. Internal genes were identified, with 4 strains had 6+ 2 constitution, 1 strain had 5+ 3 constitution. Comparing with the wild type virus, HA and NA genes of the 5 strains had no mutation. HA titer of reassortant strains was above 1 024. HI titer of the selected NO.12 reassortment strain reached 5 120, and two-way test was passed. The yield of reassortant strain was 64 times that of the wild type strain. Conclusions A circulating influenza A (H1N1) strain of influenza A (2015—2016) was successfully prepared in China and laid the foundation for vaccine storage and disease prevention and control.

Objective To analyze the phenotypic characteristics of antiviral-resistant influenza B viruses circulating in mainland China and to analyze the susceptibility of influenza B viruses to neuraminidase inhibitors ( NAIs) . Methods Antiviral-resistant phenotyping test was performed to analyze the NAI suscep-tibility of 1 386 influenza B viruses isolated in mainland China from April 2014 to March 2015, including the test of susceptibility to oseltamivir and zanamivir. Results All of the 94 B-Victoria lineage viruses were sensitive to oseltamivir and zanamivir. Of all 1 292 B-Yamagata lineage viruses tested, 1 virus showed re-duced sensitivity to oseltamivir with NA gene containing I221T amino acid mutation, 10 viruses showed re-duced sensitivity to zanamivir with 4 having D197N amino acid mutation in NA gene, 3 viruses showed re-duced sensitivity to both oseltamivir and zanamivir with NA gene possessing D197N amino acid mutation and 1 virus carrying the A245T amino acid mutation in NA gene showed reduced sensitivity to oseltamivir and highly reduced sensitivity to zanamivir. Conclusion The majority of influenza B viruses circulating in main-land China during 2014 to 2015 were sensitive to NAIs, which indicated that NAIs could be used continually for clinical treatment of patients with influenza. Sustained monitoring of antiviral susceptibility of influenza B viruses should be emphasized for timely detection of antiviral resistant viruses and more attention should be paid to the D197N mutations in NA gene of influenza B viruses.

Objective To understand the antigenic and genetic characteristics of highly pathogenic avian influenza H5N1 viruses isolated from poultry related environments of surrounding areas of Qinghai Lake.Methods Poultry and wild birds related environments from surrounding areas of Qinghai lake were collected and handled.Viruses were isolated with embryonated chicken eggs.The virus subtyping was performed by real-time RT-PCR.The highly pathogenic avian influenza H5N1 viruses were chosen to conduct next generation sequencing and hemagglutination inhibition ( HI) assay.Results In total of 19 H5N1 virus strains were isolated, they were all from poultry related environments and collected in January-March of year 2013.Seven representative highly pathogenic avian influenza H5N1 viruses were sequenced and analyzed for antigenicity.According to phylogenetic analysis of HA gene, 6 viruses were belong to clade 2.3.2.1c except A/Environment/Qinghai/XN02032/2013 which belongs to clade 7.2.Antigenic analysis showed that 6 viruses were antigenically similar to A/Barn swallow/Hong Kong/D10-1161/2010, while A/Environment/Qinghai/XN02032/2013 presented low reaction with all reference antisera.Conclusions The existence of highly pathogenic avian influenza H5N1viruses in poultry related environments of surrounding areas of Qinghai Lake was confirmed.The status of highly pathogenic avian influenza H5N1 viruses with evolution diversity emphasized the necessary of strengthening the avian influenza surveillance in these areas.