Trend analysis of hemagglutinin and ovalbumin content for influenza virus split vaccines in 2021 / 中国生物制品学杂志

@#Objective To analyze the trend of the hemagglutinin(HA) and ovalbumin contents in the lot release of influenza virus split vaccines in 2021,and evaluate the quality and quality control level of the vaccines.Methods The HA and ovalbumin content data of influenza virus split vaccines from two domestic enterprises in 2021 were collected and collated. The mean value and standard deviation were calculated according to the first 40 batches of data of the enterprise in the year,and the warning limit and action limit were established. The trend analysis of the above indexes was carried out to evaluate the stability and consistency of the product quality of the enterprise. Statistical data comparison and consistency analysis were made between the test results of the batch inspected by the lot release institution and the results of the enterprise.Results Through the retrospective data analysis of quadrivalent influenza virus split vaccines from two vaccine enterprises A and B,it was found that the content of H1N1 subtype HA and ovalbumin in the two enterprises and the content of Bv HA in the B enterprise had out of trend(OOT)situations,while the trend of other items was stable. The results of paired student's t test or Wilcoxon signed-rank test of the samples inspected by the lot release institution showed that except Bv subtype HA(t = 1. 094 and 0. 742 respectively)and ovalbumin(w =-64 and 36 respectively)contents showed no statistically significant difference(P > 0. 05),the HA contents of H1N1(t = 3. 862,w = 232),H3N2(t = 8. 225 and3. 473 respectively)and By(t = 5. 616 and 4. 934 respectively)of the two enterprises had significant differences(P <0. 05). The results of enterprises were generally higher than the lot release institution. Bland-Altman test analysis found that the consistency between the test data of enterprise A's HA content and the data of the lot release institution was better than that of enterprise B.Conclusion The stability and consistency of data trends of active ingredients and main impurity ingredients of quadrivalent influenza virus split vaccine batches in 2021 were generally good. The trend analysis can identify potential problems in vaccine production,and enterprises should carefully implement trend analysis and effectively monitor the product quality of vaccines.

Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Genetic characteristics of influenza A H3N2 virus influenza season in Xiangyang City in 2022-2023 / 公共卫生与预防医学

Objective To analyze the prevalence and genetic characteristics of influenza A(H3N2) viruses in the city of Xiangyang in 2022-2023, and to provide a scientific basis for predicting the epidemic and mutation of influenza virus. Methods Throat swab specimens of the influenza like cases were collected from national influenza monitoring sentinel hospitals in Xiangyang every week. RNA was extracted from the specimens for influenza diagnosing using real-time RT-PCR．Viruses were isolated from H3N2 positive specimens, and HA and NA genes were amplified and sequenced．3D modeling analyses were conducted. Results The gene phylogenetic tree showed that the H3N2 isolates in 2022-2023 belonged to 3C.2a1b.2a1 and 3C.2a1b.2a2 branches, respectively. The A(H3N2) influenza virus strains all had amino acid point mutation sites on important antigenic determinants of HA protein. The epitope mutations of the 2022 A(H3N2) strain mainly occurred in regions B, C, and D. The epitope mutations of the A(H3N2) strain in 2023 mainly occurred in regions C and D. Different glycosylation sites of HA gene were found in 2022-2023 strains. No variation was found in key amino acid sites associated with neuraminidase inhibitor resistance. The difference of overall structure was not obvious in the three-dimensional simulation structure diagram. Conclusion The A(H3N2) influenza strains isolated in this study have shown antigenic drift, especially the mutation of HA, which may affect the protective effect of the vaccine on the local population and lead to influenza epidemic. The variations of HA and NA suggest that close attention should be paid to the epidemic and genetic variation of H3N2 subtype influenza virus, to provide a scientific basis for the selection of influenza virus vaccine strains and the prevention and control of influenza.

Evaluation of molecular point-of-care testing for rapid diagnosis of influenza A virus and influenza B virus / 国际检验医学杂志

Objective To evaluate the performance of two molecular point-of-care testing(POCT)prod-ucts in the diagnosis of influenza A virus(Flu A)and influenza B virus(Flu B)of clinical samples,and pre-liminarily evaluate the clinical diagnostic value of the changes of infection-related indicators in peripheral blood.Methods A total of 491 oropharyngeal swabs from patients with influenza-like symptoms who were treated in the hospital were recruited into this study from November 1,2019 to June 30,2023.These swabs were collected using reverse transcription real-time quantitative fluorescent polymerase chain reaction(RT-qPCR),and two POCT molecular products,XpertTM Xpress Flu/RSV and EasyNAT? Flu Assay,respectively.The diagnostic performance of two POCT molecular products was analyzed using RT-qPCR reaction as a standard.According to the results of RT-qPCR method,the subjects were divided into Flu A positive group,Flu B positive group and negative group(both Flu A and Flu B were negative).The levels of indicators in pe-ripheral blood of the three groups were compared to evaluate the value of these indicators in the clinical diag-nosis of Flu A and Flu B.Results Among the 491 patient specimens,the XpertTM Xpress Flu/RSV assay showed the sensitivity for Flu A was 96.88%,and the specificity was 99.75%,and the sensitivity for Flu B was 100.00%,and the specificity was 100.00%.EasyNAT? Flu Assay assay showed the sensitivity for Flu A was 94.79%,and the specificity was 96.81%,and the sensitivity for Flu B was 100.00%,and the specificity was 100.00%.And two POCT molecular methods performed well consistency(Kappa value was 0.974).There was no significant difference in the levels of C-reactive protein and serum amyloid A among the negative group,Flu A positive group,and Flu B positive group(P>0.05).But the levels of white blood cell count in the negative group were higher than those in the Flu A positive group and Flu B positive group(P<0.01).Conclusion In this paper,two typical molecular POCT products are studied.Their sensitivity and specificity are highly consistent with the results of RT-qPCR.Molecular POCT products have the advantages of flexibil-ity and rapidity,which are of great value for the improvement of clinical diagnosis and treatment.Molecular detection combined with peripheral blood infection related indicators is helpful for the early diagnosis of influ-enza virus infectious diseases.

Intranasal immunization with single-dose vaccine based on recombinant influenza virus H1N1 expressing the extracellular domain of respiratory syncytial virus G protein induces robust immunity and protection in mice / 中华微生物学和免疫学杂志

Objective:To construct a novel respiratory syncytial virus (RSV) vaccine based on a recombinant influenza virus vector and evaluate its immune protective effects in mice.Methods:A recombinant H1N1 influenza A virus (IAV) expressing the extracellular domain (Gecto) of RSV A2 G protein was constructed and rescued, named as PR8NAGecto/WSN. After in vitro verification of the Gecto expression and PR8NAGecto/WSN growth kinetics, a single dose of PR8NAGecto/WSN was used to immunize BALB/c mice through intranasal administration to evaluate the efficacy of PR8NAGecto/WSN by assessing humoral (IgG, neutralizing antibody), mucosal (IgA) and cellular immunity (IFN-γ ELISPOT). Four weeks after immunization, the mice were challenged with RSV A2 or RSV B9320 to evaluate the protective effects of PR8NAGecto/WSN by analyzing mouse body weight changes, lung tissue virus titers and pathological changes. Results:A single-dose intranasal immunization with PR8NAGecto/WSN induced robust humoral, mucosal and cellular immunity in mice. Moreover, the mice in the immunized group had lower lung virus loads and mild lung pathological damages following the challenge with RSV A or RSV B subtype as compared with the control group.Conclusions:A single-dose intranasal immunization with PR8NAGecto/WSN induces robust immunity and provide protection against RSV A and B challenges in mice. This study provides new ideas and reference for the development of novel mucosal vaccines against RSV.

Etiological analysis of influenza-like cases in Taizhou City， Zhejiang Province from 2013 to 2022 / 上海预防医学

ObjectiveTo analyze the etiological results of influenza-like case surveillance in Taizhou， Zhejiang Province from 2013 to 2022， to timely understand the epidemic trend of influenza viruses and the change rule of dominant virus strains， and to provide reference for the prevention and control of influenza in this region. MethodsInfluenza virus nucleic acid was detected by real-time PCR in 24 183 influenza-like cases. ResultsThe positive rate of influenza virus in 24 183 samples was 18.43%， the highest positive type was seasonal H3 （37.34%）. There was no a significant difference in positive rate between different genders （χ2=0.148， P=0.701）. There was significant difference in the positive rate among different age groups （χ2=496.626， P<0.001）， and the highest positive rate was found in the 25‒59 age group （22.56%）. The positive rate in different years was statistically significant （χ2=1 670.922， P<0.001）. The positive rate from 2013 to 2019 showed an upward trend （χ2=30.559， P<0.001）. The lowest positive rate was in 2020 （0.04%）， and the positive rate from 2021 to 2022 showed an upward trend （χ2=304.465， P<0.001）. The dominant strains were different in different monitoring years. There was a significant difference in the positive rate of influenza in different months （χ2=1 652.455， P<0.001）， and the peak of influenza was mainly concentrated in December‒March and July‒August. ConclusionFrom 2013 to 2022， the positive rate of influenza virus in Taizhou showed a wavy dynamic change， and the dominant strains were different in different years， presenting alternate epidemic characteristics. It is necessary to strengthen the etiological surveillance of influenza virus and improve the prevention and control measures with influenza vaccine.

Prokaryotic expression and purification of influenza virus globular head domain HA1 / 中国生物制品学杂志

@#Objective To express the globular head domain HA1 gene of influenza virus(IV)in prokaryotic cells,optimize the induced expression conditions,and purify the protein in order to obtain IV HA1 protein with good immunogenicity.Methods Influenza A virus[A/Saratov/CRIE-250/2017(H3N2)],Influenza A virus[A/Hebei/F076/2018(mixed)]and Influenza A virus[A/USA/LAN＿(P5)＿HA/2018(H1N1)]were truncated,and amino acid sequence of63-286 globular head domain was obtained. The genes were optimized and synthesized according to the codon commonly used in E.coli,and named 17Sa,Hebei and USA. The 17Sa point mutation gene was named as 17Sa change. The four genes were cloned into prokaryotic expression vector pET-28a(+)to construct recombinant plasmids respectively,which were transformed into E.coli BL21(DE3)competent cells and induced by IPTG. The protein expression conditions were optimized,and the protein was purified by His labeled nickel ion protein purification column.Results The 762 bp target gene was successfully inserted,and the recombinant plasmid was confirmed to be constructed correctly by double enzyme digestion(NheⅠ/XhoⅠ). The expressed recombinant proteins USA with a relative molecular mass of about 26 000,17Sa,p17Sa and Hebei with a relative molecular mass of about 28 000,showed specific binding to mouse anti-His antibody. The recombinant proteins were all expressed in the form of inclusion bodies,and the expression was highest after induction at 37 ℃ for 8 h. The purified recombinant proteins 17Sa,17Sa change,Hebei and USA had a purity of 90%,85%,95% and 80%,respectively.Conclusion The target protein was successfully expressed and purified by prokaryotic expression system with good reactivity,which lays a foundation of the detection of influenza antibody and the development of new vaccines.

Recent advances in small-molecule inhibitors targeting influenza virus RNA-dependent RNA polymerase / 药学学报

Influenza virus causes serious threat to human life and health. Due to the inherent high variability of influenza virus, clinically resistant mutant strains of currently approved anti-influenza virus drugs have emerged. Therefore, it is urgent to develop antiviral drugs with new targets or mechanisms of action. RNA-dependent RNA polymerase is directly responsible for viral RNA transcription and replication, and plays key roles in the viral life cycle, which is considered an important target of anti-influenza drug design. From the point of view of medicinal chemistry, this review summarizes current advances in diverse small-molecule inhibitors targeting influenza virus RNA-dependent RNA polymerase, hoping to provide valuable reference for development of novel antiviral drugs.

Analysis of drug resistance gene variation of influenza A（H1N1） pdm09 virus in Huzhou City，Zhejiang Province / 上海预防医学

ObjectiveTo investigate the mutation and genetic evolution of drug resistance gene of A（H1N1） pdm09 influenza pandemic strain in 2023 in Huzhou City， Zhejiang Province. MethodsRespiratory tract specimens from 2 influenza monitoring hospitals were collected forA（H1N1） pdm09 influenza virus nucleic acid detection. Positive specimens were inoculated with MDCK cells for influenza virus isolation and sequencing. DNA Star 7.1 software and Mega 4.0 software were used to analyze the neuraminidase （NA） enzyme active site and the amino acid sites related to drug resistance in M2 protein. ResultsNucleotide homology and amino acid homology of NA between the isolated and the vaccine strains were 98.87%‒99.22% and 98.94%‒99.36%， respectively. The nucleotide homology range of M gene was 99.07% to 99.85%， and the amino acid homology range was 99.02%‒99.94%. The isolates and vaccine strains belong to the evolutionary clades of 6B.1A.5a.2a. The amino acids at the key sites of the enzyme activity center of NA were still highly conserved， and the 9 key amino acid sites related to NA inhibitor resistance did not change， but some mutations occurred at the non-enzyme active sites in some popular strains. The 5 amino acid sites related to drug resistance of M2 protein were not replaced， but the 31st amino acid sites changed from serine to asparagine. ConclusionThe A（H1N1） pdm09 pandemic strain in Huzhou in 2023 has high homology with the 2023‒2024 vaccine strain recommended by WHO. All endemic strains are resistant to amantadines.

Using guinea pig as a model for evaluation of equine influenza vaccine

Equine influenza is a highly contagious viral disease, specially among 1-5 years old naive horses. Vaccination is considered the best way to control the disease spread and outbreaks. Although foals are the main animal used for evaluation of equine influenza vaccines, guinea pigs were chosen as an alternative model in the present work, as they have a negligible antibody titer against equine influenza virus and are cheaper and easier to handle than foals. Five equine influenza vaccine batches were evaluated in two animal models, foals and guinea pigs, by injection of two doses/animal with 4 weeks apart using 2 mL/animal/dose and evaluation of immune responses by hemagglutination inhibition test and enzyme-linked immunosorbent assay. On the 7th week post vaccination, equine influenza antibodies titers reached maximum values of 9-10.2 and 8.7-10 hemagglutination inhibition units for foals and guinea pigs, respectively; sample/negative ratios were 0.126-0.464 and 0.128-0.445 for both animals, respectively. The use of guinea pigs as an animal model for the evaluation of equine influenza vaccines could be recommended instead of foals(AU)

La gripe equina es una enfermedad viral muy contagiosa, especialmente entre los caballos jóvenes de 1 a 5 años de edad. La vacunación se considera la mejor forma de controlar la propagación y los brotes de la enfermedad. Aunque los potros son el principal animal utilizado para la evaluación de vacunas contra la gripe equina, en el presente trabajo se eligieron cobayos como modelo alternativo, ya que tienen un título insignificante de anticuerpos contra el virus de la gripe equina y son más baratos y fáciles de manejar que los potros. Se evaluaron cinco lotes de vacunas contra la gripe equina en dos modelos animales, potros y cobayos, mediante la inyección de dos dosis/animal con 4 semanas de intervalo utilizando 2 mL/animal/dosis y la evaluación de las respuestas inmunitarias mediante la prueba de inhibición de la hemaglutinación y el ensayo inmunoenzimático. En la 7ª semana posvacunación, los títulos de anticuerpos contra la gripe equina alcanzaron valores máximos de 9-10,2 y 8,7-10 unidades de inhibición de la hemaglutinación para potros y cobayos, respectivamente; las relaciones muestras/negativos fueron de 0,126-0,464 y 0,128-0,445 para ambos animales, respectivamente. Podría recomendarse el uso de cobayos como modelo animal para la evaluación de vacunas contra la gripe equina, en lugar de potros(AU)

Analysis of protein composition of H5N1 influenza virus inactivated vaccine(MDCK cells) bulks / 中国生物制品学杂志

@#ObjectiveTo analyze the protein components in the bulks of H5N1 inactivated influenza virus vaccine(MDCK cells),providing experimental basis for the improvement of the quality control method and the development of the vaccine.MethodsThe H5N1 influenza virus strain was inoculated into MDCK cells. After culturing the virus for 48 h,the virus liquid was harvested,and the original liquid sample was obtained by clarification and ultrafiltration concentration,β-propiolactone inactivation,and two-step chromatography purification with Capto Q and Sephorase 4FF. Morphology of virus particles in the sample was analyzed by transmission electron microscopy,while hemagglutinin identification of the virus bulk by single radial immunodiffusion(SRID)assay,purity by high performance liquid chromatography,and protein electrophoresis by gradient SDS-PAGE. The protein components contained in the samples were analyzed by liquid chromatography-mass spectrometry(LC/MS)combined with secondary mass spectrometry sequence determination of the recovered protein polypeptides.ResultsSpherical H5N1 influenza virus particles of 80 ～ 120 nm were observed under transmission electron microscope,showing the typical shape of influenza virus. The identification test showed that the antigenicity of the virus bulks was consistent with the virus strain. The gradient SDS-PAGE analysis showed that the virus bulk had two bands. LC/MS mass spectrometry analysis showed that H5N1 influenza virus protein was the main component in the bulk samples,including NP,M1,HA,NA and other proteins of H5N1 influenza virus.ConclusionThe protein composition of the bulk during the preparation of H5N1 influenza virus inactivated vaccine was analyzed,which provided a reference for the development and quality control of this type of vaccine.

Preliminary analysis of isolation effect of influenza virus by recombinant MDCK cells stably expressing trypsinogen / 中国生物制品学杂志

@#ObjectiveTo investigate the isolation effect of influenza virus by recombinant MDCK cells(MTY6 cells)stably expressing trypsinogen.MethodsAccording to the virus isolation method recommended by the World Health Organization(WHO)Global Influenza Surveillance Network(GISN)and the National Influenza Centers(NICs),a total of 20 throat swab specimens containing positive nucleic acid for H1N1,H3N2 and B influenza virus were isolated simultaneously using MDCK and MTY6 cells. Guinea pig red blood cells and chicken red blood cells were used for agglutination test respectively and the agglutination effects of different types of red blood cells,the positive rate of virus and the titer of hemagglutinin isolated from different cells were statistically compared.ResultsThe agglutination effect of the same virus isolate on the two types of red blood cells was different. The complete agglutination time of guinea pig red blood cells was about 2 times that of chicken red blood cells,and the deposition shape showed a ring shape. The average hemag-glutinin titer was 23. 6 ± 1. 2times that of chicken red blood cells. Under the same conditions,3 samples were negative for both types of cells,11 samples were positive for both types of cells,and the other 6 samples were negative for MDCK cells while positive for MTY6 cells. The positive rate of MTY6 cells was 30% higher than that of MDCK cells. The isolated positive samples included 8 cases of H1N1 subtype and the hemagglutinin titer of virus isolated by MTY6 cells was significantly higher than that by MDCK cells[13. 0(1. 7,23. 0)times on average]. 2 cases of H3N2 and 2 cases of B were isolated,the hemagglutinin titer of each virus isolated by MTY6 cells was 11. 3 and 32. 0 times higher than that by MDCK cells on average respectively.ConclusionIn conclusion,guinea pig red blood cells were superior to chicken red blood cells for influenza virus detection by cell isolation. Under the same conditions,MTY6 cells were more sensitive than MDCK cells for influenza virus isolation,and had the potential to be used as a high-quality cell matrix for influenza virus isolation.

Effect and mechanism of Reduning injection on influenza A virus / 药学学报

The purpose of this study is to investigate the effect of Reduning injection (RI) on influenza A virus (IAV) and its mechanism. We evaluated the cytotoxicity of RI in A549 and MDCK cells by cell counting kit-8 (CCK-8) assay. Western blot and cytopathic effect (CPE) assays were applied to test the effects of RI on viral protein, CPE and virus virulence to evaluate its inhibitory effect. The proteins level of heme oxygenase 1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), phosphorylation of P38 mitogen-activated protein kinases (MAPK) and extracellular signal-regulated kinases 1/2 (ERK1/2) were detected by Western blot. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the RNA expression of interferon-α/β (IFN-α/β). The relative luciferase reporter assay was used to analyze the promoter activity and transcriptional regulation of Nrf2. The results indicated that RI inhibited IAV-induced MDCK cytopathies in a dose-dependent manner, decreased M2 protein of influenza virus and viral titer, indicating that it has definite effect on inhibiting IAV. RI promotes the phosphorylation of P38 MAPK and ERK1/2, activates the activity of Nrf2 nuclear transcription factor, increases the expression of Nrf2 protein in the nucleus, thus up-regulates the expression of HO-1 protein, and ultimately increases the IFN-α/β mRNA level. In summary, our results demonstrated that RI inhibits the replication of IAV by activating MAPK/Nrf2/HO-1 signaling pathway, revealing a new mechanism of RI against influenza virus, and providing theoretical basis for clinical treatment of influenza virus.

Characterization of Effective Components in Guizhi Jia Gegentang for Intervention of Influenza Virus Pneumonia Based on Serum Pharmacochemistry of Traditional Chinese Medicine / 中国实验方剂学杂志

ObjectiveTo characterize the efficacy components of Guizhi Jia Gegentang（GGT） in intervening influenza virus pneumonia by ultra-performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Exactive Orbitrap MS）. MethodBALB/c mice were randomly divided into normal group and GGT group（36 g·kg-1·d-1） with six mice in each group. GGT group was continuously administered GGT extract for 5 d， while the normal group was administered an equal amount of ultrapure water. Serum and lung tissue were collected after administration， and UPLC-Q-Exactive Orbitrap MS was used to characterize the prototypical and metabolic components of GGT in serum and lung tissue of mice. The components existed simultaneously in the serum and lung tissue of mice from the GGT group were defined as its functional components， and the targets of efficacy components were searched by SwissTargetPrediction database， and GeneCards database was used to query the target of influenza virus pneumonia， and then the intersection was taken to obtain potential targets of GGT for intervening in the disease. Protein-protein interaction（PPI） network analysis of potential targets was performed by STRING database， and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis on potential targets was performed by Metascape. ResultA total of 29 prototypical components and 28 metabolic components of GGT were detected in the drug-containing serum of mice， of which 11 prototypical components and 4 metabolic components were detected in the lung tissue of mice. The main metabolic pathways included reduction， hydroxylation， methylation， glucuronidation and sulfation. The results of PPI network and KEGG analysis showed that these functional components may act through their effects on targets such as albumin（ALB）， epidermal growth factor receptor（EGFR）， steroid receptor coactivator（SRC）， Toll-like receptor 4（TLR4）， nuclear transcription factor（NF）-κB and adhesion junction. ConclusionThe 11 prototypical components and 4 metabolites present simultaneously in the drug-containing serum and lung tissue of mice may be the potential therapeutic components of GGT in interfering with influenza viral pneumonia， and act through interfering with inflammatory metabolic pathways. This study can provide a reference for the mechanism study of GGT in the treatment of influenza viral pneumonia.

Research progress of respiratory virus infection associated acute necrotizing encephalopathy / 中华实用儿科临床杂志

Acute necrotizing encephalopathy (ANE) is a subtype of acute encephalopathy presented with disturbance of consciousness and symmetric bilateral thalamic necrosis in neuroradiology.Patients with ANE had a high mortality or severe neurological sequela.ANE usually secondary to virus infectious disease, in which influenza is a common etiology.During the 3 years of the worldwide pandemic of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection, ANE has become a severe complication and cause of death in children, which has aroused much concern.Here is a review of the research progress of epidemiology, pathogenesis, diagnosis, treatments and prognosis of ANE, in order to improve the knowledge of clinicians on this disease.

Preparation and immunogenicity evaluation of recombinant influenza hemagglutinin trimer vaccine / 中华微生物学和免疫学杂志

Objective:To prepare a recombinant hemagglutinin trimer (HA-Tri) vaccine against influenza viruses and to study its immunogenicity in a mouse model.Methods:A stable CHO cell line that could express HA-Tri was constructed. Western blot, single radial immunodiffusion, protein particle size detection and N-glycosylation site analysis were performed for qualitative and quantitative analysis of the recombinant protein. According to the different treatment conditions such as dosage and adjuvant, BALB/c mice were divided into 11 groups and subjected to consistent immunization procedures. Serum neutralizing antibody titers were measured on 56 d after the first immunization to evaluate the immunogenicity of HA-Tri.Results:The constructed CHO cells could secret and express HA-Tri proteins. The HA-Tri proteins were biologically active and capable of forming precipitation rings in the single radial immunodiffusion. The particle size of HA-Tri was approximately 18.79 nm and 10 N-glycosylation sites were detected, including high mannose, complex glycoforms and heterozygous glycoforms. After prime-boost immunization, there was no statistically significant difference in the titers of neutralizing antibodies induced in mice by 3.75 μg of HA-Tri in combination with RFH01 adjuvant and 15 μg of monovalent vaccine stock solution ( P=0.431 2, U=36). Serum antibody titers in the HA-Tri+ RFH01 groups were higher than those in the corresponding HA-Tri groups without RFH01 adjuvant, and the highest titer was induced in the 15 μg HA-Tri+ RFH01 group, which was 1 280. Conclusions:The recombinant HA-Tri protein was successfully prepared. HA-Tri in combination with RFH01 adjuvant could induce humoral immune responses against influenza viruses in BALB/c mice, which would provide reference for the development of influenza virus recombinant subunit vaccines.

Purification of H5N1 influenza virus by different chromatography media / 中华微生物学和免疫学杂志

Objective:To purify H5N1 influenza virus concentrate prepared by MDCK cells with a new mixed-mode chromatography medium Capto Core700 and the traditional medium Sepharose 4FF, and to compare the separation and purification efficacy of the two media.Methods:Capto Core700 and Sepharose 4FF were used to purify inactivated H5N1 influenza virus concentrate. The morphology of virus particles in different samples was then observed under a transmission electron microscope. Single radial immunodiffusion (SRID), Folin-Phenol (Lowry) method, double-antibody sandwich ELISA and qPCR were used to detect hemagglutinin, total protein, host cell protein (HCP) and host cell DNA (HCD) before and after purification. The recovery rate of virus antigen and the removal rate of impurities were calculated. The immunogenicity of the viruses purified with different media was analyzed using animal experiments. Difference in the purification efficacy of the two chromatography media was analyzed by t-test. Results:H5N1 influenza viruses purified by Capto Core700 or Sepharose 4FF showed the typical influenza virus morphology under transmission electron microscope. There was no significant difference in the recovery rate of hemagglutinin between the two chromatography media ( P>0.05), but compared with Sepharose 4FF, Capto Core700 had a higher removal rate of impurities (total protein, HCP, HCD) and the difference was statistically significant ( P<0.05). Animal experiments showed that the viruses purified by the two chromatography media had good immunogenicity. Conclusions:Compared with Sepharose 4FF chromatography medium, Capto Core700 could more effectively remove process-related impurities such as HCP, HCD and total protein without affecting the recovery rate of viral antigen. This study provided reference for the development of purification technology in the production of H5N1 influenza virus vaccine in MDCK cells.

Immunogenicity of quadrivalent influenza virus subunit vaccine combined with RFH01 adjuvant in mice / 中华微生物学和免疫学杂志

Objective:To evaluate the immunogenicity of a quadrivalent subunit vaccine combined with RFH01 adjuvant in a mouse model.Methods:Identification tests were performed on four monovalent influenza virus subunit vaccine stock solutions according to the methods described in Part 3 of the Chinese Pharmacopoeia 2020 Edition. In the study of the quadrivalent subunit vaccine combined with RFH01 adjuvant, 460 female BALB/c mice (6-8 weeks old) were randomly divided into 46 groups including experimental groups, vaccine control group, negative control group and blank group with 10 mice in each group. In the study of the quadrivalent subunit vaccine in old and young mice, 80 female 10-month-old and 80 female 10-week-old BALB/c mice were randomly divided into 16 groups ( n=10) including monovalent influenza virus vaccine group, quadrivalent subunit vaccine group, quadrivalent subunit vaccine+ RFH01 adjuvant group, chicken embryo quadrivalent split vaccine control group and PBS group. All mice were immunized by intramuscular injection. At 21 d after the primary immunization, a booster immunization was conducted using the same strategy. Blood samples were collected at 21 d and 42 d after the primary immunization for serum separation. Haemagglutination inhibition (HI) test was performed to detect the antibody levels in mouse serum samples. Results:After the booster immunization, the positive conversion rates in all vaccine+ RFH01 adjuvant groups reached 100%, and the geometric mean titers (GMTs) of serum antibodies were significantly higher than those of the vaccine groups without RFH01 adjuvant. There were significant differences in serum antibody titers between the monovalent/quadrivalent subunit vaccine groups with and without RFH01 adjuvant. After the booster immunization, the titers of serum antibodies against H1N1, H3N2, B/Victoria and B/Yamagata in the 10-week-old mice were significantly higher than those in the 10-month-old mice.Conclusions:The monovalent and quadrivalent influenza virus vaccines in combination with RFH01 adjuvant could elicit higher antibody titers in young (6-10 weeks old) and old (10 months old) mice, showing good immunogenicity.

Establishment of an in vivo neutralization model based on H5N1 pseudovirus / 中华微生物学和免疫学杂志

Objective:To establish an in vivo infection model of H5N1 pseudovirus and to detect the neutralizing activity of FHA3 antibody using this model. Methods:Based on the sequence information of hemagglutinin (HA) and neuraminidase (NA) of A/Anhui/1/2005/H5N1 strain, two recombinant plasmids of pcDNA3.1-HA5 and pcDNA3.1-NA1 were constructed. The two plasmids and plasmid pNL4-3.Luc.R-E- were co-transfected into 293T cells to prepare H5N1 pseudovirus supernatant. The morphology of pseudovirus particles in the supernatant was observed by electron microscopy. MDCK cells were infected with the pseudovirus supernatant and the virus titer was detected. BALB/c mice were injected with the pseudovirus supernatant by intraperitoneal injection and subjected to bioluminescence imaging at 2, 5, 8, and 12 d after infection to detect the pseudovirus infection in vivo. The functional activity of FHA3 antibody in vivo was evaluated using the established mouse infection model. Results:The recombinant plasmids pcDNA3.1-HA5 and pcDNA3.1-NA1 were correctly constructed and could be used to prepare pseudovirus supernatants of high titer by co-transfecting 293T cells with the plasmid pNL4-3.Luc.R-E-. The virus particles were round under electron microscope. H5N1 pseudovirus-infected mice exhibits strong fluorescence signals, which were attenuated by FHA3 treatment before challenge.Conclusions:The in vivo infection model of H5N1 pseudovirus was successfully constructed and FHA3 antibody was proved to be protective against the pseudovirus infection.

Research progress in antigenic evolution of influenza A (H3N2) virus / 中华微生物学和免疫学杂志

Influenza viruses are common pathogens causing respiratory infections in humans. Among the four seasonal influenza viruses, influenza A virus H3N2 has become the leading cause of seasonal influenza illness and death, posing a great threat to public health and the economy. Since it first emerged and caused a pandemic in 1968, H3N2 has been circulating repeatedly in human beings and continually evades host immune attack by antigenic drift, resulting in a decrease in vaccine efficacy. In this paper, the antigenic evolution of influenza A virus H3N2, the impact of antigenic evolution on the selection of vaccine strains and some models for predicting the evolution of influenza viruses were analyzed and reviewed, which paved the road for understanding the antigenic evolution of influenza virus and vaccine development.