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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.
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.
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@#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.
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@#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.
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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.
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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.
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Carnosic acid (CA) is the main phenolic diterpenoid active ingredient in plants such as rosemary and sage, and has antiviral, antioxidant, anti-inflammatory effects and so on, however, its antiviral activity against influenza virus infections was not reported. In this study, antiviral activities against influenza A virus infections of three main bioactive ingredients from rosemary, including rosmarinic acid, CA and ursolic acid, were evaluated using virus titer titration assay, and CA showed remarkable inhibition on influenza H5N1 replication in A549 cells. The antiviral activity of CA was further confirmed and its mechanism of action was investigated using the indirect immunofluorescence assay (IFA), Western blot and real-time fluorescence quantification polymerase chain reaction (qRT-PCR). The results showed that the 50% effective concentration (EC50) of CA against influenza H5N1 in A549 cells and MDCK cells were 4.30 and 3.64 μmol·L-1, respectively. Meanwhile, CA also showed inhibition on influenza virus 2009panH1N1 (EC50: 10.1 μmol·L-1) and H3N2 (EC50: 12.8 μmol·L-1) replications in A549 cells. Mechanistic studies showed that antiviral activity of CA is related to its induction of heme oxygenase-1 (HO-1) in A549 cells and suppression on production of reactive oxygen in H5N1-infected cells.
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@#Abstract：In recent years，the outbreak and prevalence of respiratory infectious diseases in the world seriously endanger human health，among which the respiratory infectious diseases caused by viral infection account for a large proportion. The use of vaccines and common antiviral drugs is an effective way to fight viral infection，but there are also problems such as lag and drug resistance. Monoclonal antibodies against respiratory viral infections provide a new strategy for clinical treatment. This paper reviews the development of monoclonal antibody against respiratory virus and its application in respiratory viral infectious diseases. Keywords：Respiratory viral infectious diseases；Respiratory syncytial virus（RSV）；Influenza virus（IFV）；Coronavirus （CoV）；Monoclonal antibody
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ObjectiveTo establish a rapid screening method for influenza virus neuraminidase（NA） inhibitors sourced from Chinese medicines based on fluorescence detection. MethodThe method was constructed based on the principle that after the reaction of the test sample and a certain amount of NA， the activity of some NA will be inhibited by the test sample， and the NA that is still active after the addition of the substrate can generate fluorescence at a specific wavelength when combined with the fluorescent substrate， and the inhibition rate of the test sample on NA was calculated according to the measured fluorescence intensity， so as to evaluate the in vitro inhibitory activity of the test sample on NA. A total of 49 high-purity chemical components from 12 Chinese medicines were used to evaluate the in vitro anti-NA activity by the established method. The theoretical calculated values of binding energy and inhibition constant after docking between the NA protein receptor and the test sample were used to prove the accuracy of the experimental results. The established method was applied to detect the in vitro NA inhibitory activity of different batches of Banlangen granules and Kangbingdu granules， so as to evaluate the quality consistency among different batches of samples. ResultThe methodological examination results showed that the method had good accuracy and repeatability. The screening results of 49 components showed that 22 of them had strong in vitro inhibitory activity against NA than peramivir ［half inhibitory concentration（IC50） was 131.2 μmol·L-1］， such as schaftoside， isoorientin， chebulinic acid， menthone and isoschaftoside. The inhibitory activity of the remaining 27 components was weaker than that of peramivir. The molecular docking results showed that the theoretical calculation results of binding energies and inhibition constants of most compounds were basically consistent with the experimental results. The test results of the inhibitory activity of 12 batches of Banlangen granules on NA showed that the quality consistency among samples A1， A2， B2， C1， C2， E2 and F2 was good. The analysis results of the inhibitory activity of 9 batches of Kangbingdu granules produced by the same manufacturer on NA showed that the inhibitory rates of samples K1 to K9 were 37.68%， 36.18%， 31.37%， 33.98%， 40.36%， 33.76%， 40.69%， 41.08%， 40.06% when the concentration of 0.02 g·mL-1， and the average inhibitory rate was 37.24%. ConclusionIn this paper， we successfully established an analytical method that can be used to rapidly evaluate whether Chinese medicines （derived from chemical components of traditional Chinese medicine or proprietary Chinese medicines） have in vitro anti-NA activity， which can be a powerful supplement to the existing screening methods for influenza virus NA inhibitors. And this method was used to screen 22 compounds from 12 Chinese medicines with good in vitro inhibitory activity against NA， which can provide candidate compounds for the development of anti-influenza small molecule drugs.
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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.
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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.
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@#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.
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@#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.
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ObjectiveTo determine the prevalence of avian influenza viruses in the external environment of poultry in Anqing City， Anhui Province， and provide scientific evidence for prevention and control of animal-derived influenza in humans. MethodsA total of 28 farmers’ markets/farms in 10 counties （cities， districts） of Anqing City， Anhui Province， were selected as surveillance sites by simple random sampling strategy. Poultry faeces and other related samples were collected for 6 consecutive weeks. Real-time fluorescence quantitative PCR was used to examine the nucleic acids of influenza A virus. Subtypes H5， H7， and H9 of avian influenza virus were further tested in the positive samples. ResultsA total of 426 specimens were collected， among which 113 tested positive with a positive rate of 26.53%. Among the positive specimens， 104 were determined to be subtype H9， accounting for 92.04%. It did not significantly differ in the positive rate between the main and non-main urban areas （χ2<0.01， P>0.05） or among the specimens collected in different weeks （χ2=7.57， P>0.05）. However， it significantly differed in the positive rate among the specimens collected in the third week and other weeks （χ2=6.89， P<0.05）. Furthermore， among the different sampling sites， farms had the highest positive rate of 46.67%. Among the specimens from different sources， the surface-coated specimens from poultry cages had the highest positive rate of 34.78%. ConclusionAvian influenza viruses are prevalent in the external environment of poultry in Anqing City. It warrants strengthening the surveillance and risk assessment to reduce the virus transmission in the external environment and risk of human infection with animal-derived influenza.
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Hemagglutinin and neuraminidase, two important glycoproteins on the surface of influenza virus, play a considerable role in the entry and release stage of the viral life cycle, respectively. With in-depth investigation of influenza virus glycoproteins and the continuous innovation of drug discovery strategies, a new generation of glycoproteins inhibitors have been continuously discovered. From the point of view of medicinal chemistry, this review summarizes the current advances in seeking small-molecule inhibitors targeting influenza virus glycoproteins, hoping to provide valuable guidance for future development of novel antiviral drugs.
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@#ObjectiveTo investigate the effect of activation of RIG-Ⅰsignaling pathway by leucine rich repeat containing23(LRRC23)on replication of influenza virus.MethodsOverexpression and knock-down of LRRC23 were performed in A549 cells to investigate its effect on influenza virus replication. A549 cells were transfected with pcLRRC23 plasmid or siLRRC23(small interfering LRRC23)for 24 h and then infected with influenza virus A/jingfang/1/86(H1N1). The virus titer and HA protein expression level in the cell supernatant were determined by plaque assay and ELISA respectively.The expression of LRRC23,RIG-Ⅰ,MAVS,M1 and HA at gene and protein levels were determined by qRT-PCR and Western blot respectively. The interactions between LRRC23 and RIG-Ⅰwere analyzed by co-immunopre-cipitation(Co-IP)and immunofluorescence assay(IFA). IFN-β-luc and NF-κB-luc activities were determined by dual-luciferase reporter assay.ResultsThe LRRC23 overexpression significantly decreased the influenza virus titer and inhibited the expression of HA protein in the supernatant of A549 cells,while enhanced the NF-κB and IFNβ activations by activation of RIG-Ⅰ-MAVS signaling pathway,resulting in the inhibition of expressions of M1 gene and HA protein. Conversely,the knock-down of LRRC23 increased the protein expression level of HA in the supernatant of A549 cells,up-regulated the relative expression level of M1 gene and down-regulated those of RIG-ⅠmRNA and MAVS mRNA.ConclusionLRRC23 plays an essential role in innate antiviral response by inhibiting influenza virus replication through activation of RIG-Ⅰsignaling pathway.
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@#Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc). Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay. A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200 μL per mouse. Blood samples were collected at 5,60,120,240 and 360 min after injection. Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software. Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6. 67 × 10~4[1/（M*s）]. The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1. TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly. The half-life of TAT-ScFv-mFc in mice was 212 min. Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.
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@#Influenza vaccination is the most effective route to prevent influenza virus transmission,and adding adjuvant to influenza vaccines not only enhances the body immune response but also saves the antigen dose.In addition to the classic aluminum adjuvant,MF59 adjuvant is the second one on the market and has been widely used as adjuvant in human vaccine,which not only has an ability to induce antibody equivalent to that of aluminum adjuvant,but can induce cellular immune response,recruit immune cells,and improve the effectiveness of vaccine.In this review,the mechanism of action and current application progress of MF59 adjuvant in influenza vaccine was described,and the further development of MF59adjuvant research was put forward.
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@#Objective To evaluate the passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells(sMDCK)of serum-free suspension culture.Methods H5N1(NIBRG-14) influenza virus working-bank vaccine strains were passed 15 consecutive times in sMDCK cells.The 8-segment nucleotide sequences(HA,NA,M,NP,NS,PA, PB1 and PB2 genes) of the main-bank,working-bank,virus of P1,P2,P3,P5,P10 and P15 generations were detected for genetic stability by second and first generation sequencing.The stability of amino acid sequences of hemagglutinin(HA)and neuraminidase(NA),the main antigens of the working-bank,P5 and P15 generation viruses,were evaluated by using peptide coverage as indicators;Influenza vaccine was prepared with working-bank,P5 and P15 generation viruses,with which the female BALB/c mice were immunized i.m.with 10 in each group,15 μg HA per mouse,and boosted 28 d later at the same dosage and route.At 28,42 and 56 d after the primary immunization,the mice were detected for the titer of neutralizing antibody in serum to evaluate the stability of immunogenicity.Results No segment insertion or deletion was detected in each generation of influenza virus,and the nucleotide sequence was completely consistent with the main-bank;Single nucleotide polymorphism(SNP) mutations did not occur in the main-bank,working-bank,P1,P2,P3,P5 and P10 generations of viruses,while the possibility of SNP mutation showed in many gene loci of P15 generation virus,with heterozygous SNP accounting for 91.62%.The coverage rate of HA and NA protein peptides of P5 and P15 generation viruses ranged from96.7% to 100%.There was no significant difference in serum neutralizing antibody titer of mice in the working-bank,P5 and P15 groups(H=2.253,2.029 and 1.408,P=0.324,0.363 and 0.495,respectively) at 28,42 and 56 d after the first immunization.Conclusion H5N1(NIBRG-14) influenza virus vaccine strain has good genetic stability in sMDCK cells,which is expected to be used in the production of sMDCK cell matrix pandemic influenza vaccine.
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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.
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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.