Thermal stability of neuraminidase in influenza vaccine / 中国生物制品学杂志

@#Abstract：Objective To analyze the stabilities of neuraminidase（NA）in influenza vaccine at different temperatures and provide a reference for further complete understanding of overall shelf life of vaccines. Methods Monovalent bulks of influenza H1N1，H3N2 and B vaccines were stored at 4（low temperature），25（room temperature）and 37 ℃（changed temperature）for 0. 5，2，7，24 and 48 h separately，using that at 100 ℃（extreme temperature）for 1 h as control，and determined for NA activity by enzyme⁃linked lectin method. Results The NA activities of influenza H1N1 vaccines stored at 25 and 37 ℃ decreased significantly with the increasing of time. No significant decreases were observed in H3N2 and B vaccines even after storage at two non⁃storage temperatures for 48 h. However，all the NA activities of three vaccines decreased at 100 ℃. Conclusion Both H3N2 and B vaccines showed high stability at abnormal storage temperatures not more than 37 ℃，while H1N1 vaccine was relatively sensitive to the temperature for storage.

Establishment and Application of Rapid Screening Method for Influenza Virus Neuraminidase Inhibitors Sourced from Traditional Chinese Medicine Based on Fluorescence Detection / 中国实验方剂学杂志

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.

Recent advances in small-molecule inhibitors targeting influenza virus glycoproteins / 药学学报

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.

Genomic characterization of seven reassortant influenza B viruses in Hangzhou, China / 中华微生物学和免疫学杂志

Objective:To analyze the prevalence of influenza B virus in Hangzhou from 2014 to 2020 and the genetic evolution of seven reassortant strains of influenza B virus.Methods:Influenza viruses were isolated from throat swabs collected from 16 943 patients with influenza-like illness in Hangzhou from January 2014 to December 2020. The subtypes of influenza viruses were identified by real-time RT-PCR. Eight genes ( PB2, PB1, PA, HA, NP, NA, MP and NS) of influenza B viruses were amplified with specific primers and then analyzed with nanopore sequencing and phylogenetic analysis. Results:From January 2014 to December 2020, there were 1 090 influenza B virus-positive samples, including 474 samples of Yamagata lineage and 616 samples of Victoria lineage, were identified in Hangzhou with an overall positive rate of 6.43% (1 090/16 943). Whole genomes of 228 strains of influenza B virus were obtained by nanopore sequencing and seven reassortant strains of influenza B virus were found. There were four reassortant influenza B viruses of Yamagata lineage with NA gene fragments from viruses of Victoria lineage, two strains of Yamagata lineage (H644_BY and H648_BY) with NP and NA gene fragments from Victoria lineage and one strain of Victoria lineage with PB2, PB1, PA and NS gene fragments from Yamagata lineage. Meanwhile, these seven strains possessed several mutations in the antigenic sites of HA and NA genes. Conclusions:Several rare reassortant strains of influenza B virus with epidemic potential were detected in Hangzhou from 2014 to 2020, which indicated that the traditional detection methods should be improved and more attention should be paid to the reassortant influenza B viruses and the match between epidemic and vaccine strains.

Characteristics of hemagglutinin-neuraminidase gene of human parainfluenza virus type 3 among patients with acute respiratory tract infection in Henan Province / 中华微生物学和免疫学杂志

Objective:To analyze the molecular characteristics of hemagglutinin-neuraminidase (HN) gene of human parainfluenza virus type 3 (HPIV3) among the cases with acute respiratory tract infection (ARI) in Henan Province.Methods:Nasal/throat swab samples collected from patients with severe acute respiratory tract infection (SARI) in Luohe and patients with influenza-like illness (ILI) in Zhengzhou were used in this study. HPIV nucleic acids in the samples were detected using real-time fluorescent PCR. HPIV3-positive samples were subjected to RT-PCR for the amplification of HN genes and the sequences were analyzed with Sanger method. CExpress and MEGA7.0 software were used for sequences editing, evolution tree construction and gene sequence analysis.Results:A total of 374 throat swab samples collected form ARI cases in Luohe and Zhengzhou were tested and 20 (5.3%) of them were positive for HPIV3. Eighteen HPIV3 HN gene sequences were successfully amplified and all belonged to C3 subgroups, including 16 sequences of C3f genotype and two sequences of C3a genotype. The 18 HN gene sequences shared the homology of 97.6%-100.0% in nucleotide and 99.3%-100.0% in amino acid, but the differences between them and the prototype strain Wash/47885/57 were significant. There were 12 amino acid mutations shared by them, including four function-related mutations (H295Y, I391V, D556N and I53T). There were no significant differences in the nucleotide or amino acid sequences as compared with the epidemic strain of China/BCH4210A/2014.Conclusions:The C3f and C3a branches of HPIV3 were the epidemic genotypes in Henan Province in recent years and a local circulating prevalence might be established. Continuous and in-depth monitoring of HPIV3 C3 subtype would be of great significance for the prevention and control of HPIV3-associated diseases.

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virus-induced oxidative stress

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics. Currently, neuraminidase (NA) inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration (FDA) for the prevention and treatment of influenza. Here, we show that vitisin B (VB) inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells. Reactive oxygen species (ROS), which frequently occur during viral infection, increase virus replication by activating the NF-κB signaling pathway, downmodulating glucose-6-phosphate dehydrogenase (G6PD) expression, and decreasing the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response activity. VB decreased virus-induced ROS generation by increasing G6PD expression and Nrf2 activity, and inhibiting NF-κB translocation to the nucleus through IKK dephosphorylation. In addition, VB reduced body weight loss, increased survival, decreased viral replication and the inflammatory response in the lungs of influenza A virus (IAV)-infected mice. Taken together, our results indicate that VB is a promising therapeutic candidate against IAV infection, complements existing drug limitations targeting viral NA. It modulated the intracellular ROS by G6PD, Nrf2 antioxidant response pathway, and NF-κB signaling pathway. These results demonstrate the feasibility of a multi-targeting drug strategy, providing new approaches for drug discovery against IAV infection.

Molecular characterisation of hemagglutinin and neuraminidase genes of the first highly pathogenic Avian Influenza H5N1 2.3.2.1c virus isolated from Sabah, Malaysia

@#Highly Pathogenic Avian Influenza (HPAI) is a highly contagious disease in poultry. The outbreaks can lead to flock mortality up to 100% in two to three days. In July 2018, high mortality in a commercial layer farm in Kauluan village, Sabah was reported. Samples were sent to Veterinary Research Institute Ipoh for diagnosis. Virus isolation and molecular detection is carried out simultaneously. The causative agent was then identified as AI H5N1 virus by real time reverse transcription-polymerase chain reaction (RT-PCR). The virus was then subjected for further nucleotide sequencing of full length hemagglutinin (HA) and neuraminidase (NA) gene. The PQRERRRKR/GLF motif at the HA cleavage site indicated that the isolate was of HPAI virus. Phylogenetic analysis of the HA gene showed that the isolate was belonged to the clade 2.3.2.1c virus. In the HA gene, besides the S133A substitution, the virus possesses conserved amino acid at most of the avian receptor binding sites including the glutamine (Q) and glycine (G) at position 222 and 224 respectively, indicating that the virus retains the avian-type receptor binding preference. As such, the zoonotic potential of the virus was relatively low. On the other hand, though the N154D and T156A substitution were detected in the same gene, the pandemic potential of this Sabah 2.3.2.1c virus is low in the absence of the Q222L, G224S, H103Y, N220K and T315I. A typical 20 amino acid deletion with loss of four corresponding glycosylation sites in the NA stalk region was visible. Though three NA resistance markers were detected, the virus was predicted to be sensitive to NA inhibitor. This is the first HPAI H5N1 outbreak in Sabah. The introduction of this virus into East Malaysia for the first time raised an alert alarm of the future epidemic potential. Strict farm biosecurity, continuous surveillance programme in poultry, wild birds, migratory birds; molecular epidemiology as well as risk assessment for the virus with pandemic potential are needed in dealing with emergence of new influenza virus in the country.

Preparation and identification of antibodies targeting influenza A virus N1 subtype neuraminidase / 中华微生物学和免疫学杂志

Objective:To prepare and identify a functional antibody FNA1 targeting the neuraminidase (NA) of influenza A virus N1 subtype.Methods:According to single-chain antibody fragment (scFv) sequence, the heavy chain and light chain variable region sequences of FNA1 were synthesized, and the recombinant expression plasmid pFRT-IgG1κ-FNA1 was constructed by linking the expression vector pFRT-IgG1κ. The FNA1 antibody was expressed in ExpiCHO cells and purified using affinity purification technique. The binding ability of FNA1 to the target proteins, influenza A virus N1 subtype NA antigens, was detected by ELISA. Flow cytometry was performed to analyze the binding ability of FNA1 to the NA antigens expressed on the surface of cell membrane. The in vitro activity of FNA1 against NA was evaluated by infecting 293T cells with pseudovirus. Results:Protein electrophoresis showed that FNA1 with high purity was obtained. FNA1 specifically recognized and bound to N1 subtype NA antigens in a concentration-dependent manner. FNA1 could effectively block NA activity by binding to N1 subtype NA protein expressed on the surface of cell membrane, thus inhibiting the release of packaged pseudovirus from cell surface and further inhibiting target cell infection.Conclusions:An antibody FNA1 targeting influenza A virus N1 subtype NA with in vitro functional activity was obtained.

Isolation and purification of neuraminidase from influenza vaccine and reference substance preparation / 中华微生物学和免疫学杂志

Objective:To establish a method for isolation and purification of neuraminidase from influenza vaccine and to prepare reference substance for quantitative detection of neuraminidase.Methods:Functional magnetic particles with specific affinity for neuraminidase were prepared. The method for separation and purification of neuraminidase was established based on the magnetic particles. The separation and purification conditions were optimized. The purity of neuraminidase was analyzed and the specificity was verified. The enzyme activity was determined and the protein was quantified.Results:The functional magnetic particles modified with 4-aminophenanthroline were successfully prepared and the method for isolation and purification of neuraminidase based on the magnetic particles was established. The purity of neuraminidase was 98.7%. The concentrations of neuraminidase isolated and purified from the monovalent stock solution of H1N1, H3N2, B/Victoria and B/Yamagate vaccines were 71.50, 100.58, 64.11 and 37.68 μg/ml, respectively, and the enzyme activity remained.Conclusions:The method for isolation and purification of influenza virus neuraminidase was established and the corresponding reference substance was prepared.

Drug resistance analysis of influenza virus to neuraminidase inhibitor from 2018 to 2019 in Hebei Province / 中华疾病控制杂志

Objective To analyze the resistance of influenza virus to neuraminidase inhibitors (NAI) in Hebei province during 2018-2019. Methods Virus were collected from the Hebei Influenza Surveillance Network during 2018-2019. A total of 36 confirmed influenza viruses (with 25 H1pdm09 and 11 H3N2) were selected to test resistance to oseltamivir and zanamivi with fluorescence (FL). Results All 36 influenza viruses tested were sensitive to oseltamivir and zanamivir. The median half maximal inhibitory concentration (IC50) for oseltamivir of H1pdm09 and H3N2 were of 0.50 nM (range 0.07-1.14 nM) and 0.25 nM (range 0.09-0.69 nM) respectively, while 0.29 nM (range 0.09-0.85 nM) and 0.87(range 0.17-1.81 nM) for zanamivir, all were within 10 fold IC50 of the reference virus (corresponding type). Conclusion All the tested influenza strains isolated in Hebei province during 2018-2019 were sensitive to NAI.

Molecular characteristics of the hemagglutinin 1 and neuraminidase genes of influenza B viruses isolated in Yancheng city from 2015 to 2017 / 中华疾病控制杂志

Objective To analyze the genetic characteristics of the hemagglutinin （HA) and neuraminidase （NA) genes of influenza B viruses isolated in Yancheng City from 2015 to 2017. Methods The throat swab specimens of influenza-like illness( ILI) from sentinel surveillance hospital and outbreak sites were collected and sent to Yancheng CDC for virus nucleic acids and virus isolation testing. After validation with serological tests, eighteen strains of influenza B virus isolates were selected to amplify their HA1 and NA genes through RT-PCR assay. Their molecular characteristics of the obtained viral HA1 and NA gene sequences were analyzed using bioinformation software from three aspects, including nucleic acid level, amino acid level and molecular evolution level. Results Basically, the clustering relationships and the branche patterns between HA1 and NA genes from the 18 Yancheng influenza B virus strains were similar. The Yamagata lineage strains in 2015 were distributed in the Yamagata Clade 3 branch, belonging to Phuket/3073 strains. The Victoria lineage strains in 2016-2017 were distributed in the Victoria Clade 1A branch, belonging to Brisbane/60 strains. D196N substitution was detected on HA1 protein in all of Yamagata lineage strains at 190-helix epitope; Amino acid substitutions of victoria lineage strains involved two antigenic epitopes, 117 and 129 sites of 120-loop epitope and 197 and 199 sites of 190-helix epitope. No Intra-lineage or inter-lineage rearrangements occurred in Yancheng strains. Eighteen influenza B strains had no mutations in catalytic residues and drug resistant sites of NA genes. Conclusion The Yamagata strains well matched with vaccine strain B/Phuket/3073/2013. The HA1 and NA genes of victoria lineage strains circulated in Yancheng City during 2016 to 2017 are changing gradually. The accumulation of these mutations will result in antigenic drift of victoria lineage strains and increase the mismatch of the IFV field stains with the available vaccine strains, which may reduce the protective effect of flu vaccine.

Assessment of hemagglutination activity of porcine deltacoronavirus

Drug resistence and NA gene characteristics of influenza virus A/H1N1(pdm09) studied in Shanghai during 2018-2019 / 上海预防医学

Objective A/H1N1(pdm09) viruses were the dominant strains in Shanghai during 2018-2019 influenza surveillance year.This study is to provide a scientific reference for clinical drug use by investigating the susceptibility of A/H1N1(pdm09) viruses to neuraminidase inhibitors(NAIs). Methods Sixty strains of A/H1N1(pdm09) viruses were randomly selected for testing the susceptibility and drug resistance to Oseltamivir and Zanamivir by means of neutaminidase inhibition and neuraminidase (NA) gene sequencing. Results The 60 epidemic strains all proved to be susceptible to Oseltamivir and Zanamivir and the susceptibility was not observed to be decreased or remarkably decreased.In genetic sequencing, NA was not observed to present amino acid mutation at the key sites and auxiliary sites in catalytic activity, which confirmed the results of the phototypic detection of neuraminidase inhibition. Conclusion The subtype influenza viruses A/H1N1(pdm09) circulating in Shanghai during 2018-2019 surveillance year are still sensitive to NAIs, which provides a scientific reference for clinical use of drugs.However, we monitored only a number of strains and think that the work monitoring antiviral susceptibility should be continued with the wide use of the drugs.

Diseño y evaluación de la expresión de una potencial vacuna de ADN contra el virus de la Tilapia de Lago (TiLV)

El Virus de la Tilapia del Lago (TiLV), es un patógeno causante de mortalidades masivas tanto en poblaciones de tilapias cultivadas y silvestres alrededor del mundo. El desarrollo de una vacuna efectiva contra este patógeno emergente es imperativo para prevenir pérdidas económicas. En este trabajo se diseñó y evaluó un vector de expresión como una potencial vacuna de ADN contra este virus. Inicialmente, se realizó un análisis de enhebramiento para predecir las estructuras tridimensionales y las funciones de las proteínas del TiLV. Se encontraron homologías estructurales entre las proteínas correspondientes al segmento genómico 1 y al segmento genómico 4 del TiLV, con las proteínas de ARN polimerasa dependiente de ARN del virus de la influenza B (56%) y la proteína neuraminidasa que pertenece a la cápside del virus de la influenza A (12%), respectivamente. Se insertó el producto de PCR del gen neuraminidasa viral en el vector plasmídico de expresión pCMV. Finalmente, se inyectó el constructo plasmídico en juveniles de la tilapia del Nilo Oreochromis niloticus y se midió su expresión mediante RT-PCR en tiempo real a las 8h, 16h, 24h, 72h después de la segunda inyección inmunizante. Se logró detectar expresión génica en los cuatro tiempos evaluados, con mayor expresión a las 16 horas post inyección. Estos resultados constituyen el primer paso para el desarrollo de una vacuna efectiva para la protección de los stocks de tilapias alrededor del mundo.

Tilapia Lake Virus (TiLV) is a pathogen that causes massive mortalities in both cultured and wild tilapia populations around the world. The development of an effective vaccine against this emerging pathogen is imperative to prevent economic losses. In this work an expression vector was designed and evaluated as a potential DNA vaccine against this virus. Initially, a threading analysis was done to predict the threedimensional structures and functions of the TiLV proteins. Structural homologies were found between the TiLV proteins corresponding to the genomic segment 1 and the genomic segment 4, with the RNA-dependent RNA polymerase proteins of the influenza B virus (56%) and the neuraminidase protein belonging to the influenza A virus capsid (12%), respectively. The PCR product of the viral neuraminidase gene was inserted into the expression plasmid vector pCMV. Finally, the plasmid construct was injected into juveniles of the Nile tilapia Oreochromis niloticus and its expression was measured by real time RT-PCR at 8h, 16h, 24h, and 72h after the second immunizing injection. It was possible to detect gene expression in the four evaluated times and greater expression at 16 hours post injection. These results are the first step in the development of an effective vaccine for the protection of tilapia stocks around the world.

Genetic characterisation of influenza A(H1N1)pdm09 viruses circulating in Assam, Northeast India during 2009�15

Introduction: Influenza A(H1N1)pdm09 virus, since its identification in April 2009, has continued to cause significant outbreaks of respiratory tract infections including pandemics in humans. In the course of its evolution, the virus has acquired many mutations with an ability to cause increased disease severity. A regular molecular surveillance of the virus is essential to mark the evolutionary changes that may cause a shift to the viral behavior. Materials and Methods: Samples of Throat/Nasal swabs were collected from a total of 3715 influenza-like illness cases and screened by Real-time Reverse Transcription-Polymerase Chain Reaction for influenza viruses. Nucleotide sequence analysis was done to identify changes in antigenicity of the virus strains. Results: The present study describes the molecular characteristics of influenza A(H1N1)pdm09 viruses detected in Assam of Northeast India during 2009�15. Influenza A viruses were detected in 11.4% (425/3715), of which influenza A(H1N1)pdm09 viruses were detected in 41.4% (176/425). The nucleotide sequencing of influenza A(H1N1)pdm09 viruses revealed a total of 17 and 22 amino acid substitutions in haemagglutinin (HA) and neuraminidase (NA) genes of the virus, respectively, compared to contemporary vaccine strain A/California/07/2009. The important mutations detected in HA genes of A/Assam(H1N1)pdm09 strains included E391K, K180Q and S202T. Mutation 'N248D' which has an ability to develop oseltamivir resistance was also detected in NA gene of A/Assam(H1N1)pdm09 strains. Conclusions: Regular molecular surveillance of influenza A(H1N1)pdm09 is important to monitor the viral behavior in terms of increase virulence, drug resistance pattern and emergence of novel strains.

Molecular characterization of virulence genes cctA, nanA, and fliC in Clostridium chauvoei from Rio Grande do Sul and São Paulo State, Brazil / Caracterização molecular dos genes de virulência cctA, nanA e fliC em Clostridium chauvoei isolados no Rio Grande do Sul e São Paulo, Brasil

ABSTRACT: Clostridium chauvoei toxin A (CctA), neuraminidase (NanA), and flagellin (FliC) proteins contribute to the pathogenicity of Clostridium chauvoei, the causative agent of blackleg in cattle. The aim of this study was to analyze the genetic variability of cctA, nanA, and fliC genes in C. chauvoei isolates from the Rio Grande do Sul and São Paulo state- Brazil, during different sampling periods. The presence of these genes was verified through PCR amplification and partial gene sequencing of 17 strains. Alignment of PCR amplicons combined with bioinformatics analysis was used in an attempt to study the variability across C. chauvoei solates. The similarity among the partial sequences of cctA and nanA genes was 100%. The sequencing of fliC revealed three different paralog alleles of flagellin, and two strains were seen to be polymorphic, with amino acid alterations in the predicted protein. Overall, this study indicates that strains of C. chauvoei isolated in Brazil are highly conserved with respect to the virulence factors evaluated.

RESUMO: Toxina A de Clostridium chauvoei (CctA), neuraminidase (NanA) e flagelina (FliC) são proteínas que contribuem para a patogenicidade de Clostridium chauvoei, o agente causador do carbúnculo sintomático em bovinos. O objetivo deste estudo foi analisar a variabilidade genética dos genes cctA, nanA, e fliC em C. chauvoei isolados em diferentes períodos no Rio Grande do Sul e São Paulo. A presença destes genes foi verificada pela amplificação dos produtos da PCR e sequenciamento parcial dos genes de 17 cepas. Os alinhamentos da amplificação dos produtos da PCR combinados com a análise de bioinformática foram utilizados na tentativa de avaliar a variabilidade dos genes entre os isolados de C. chauvoei. A similaridade do sequenciamento parcial dos genes cctA e nanA foi 100%. O sequenciamento do fliC revelou três alelos paralogos diferentes de flagelina e duas cepas mostraram polimorfismos, causando alterações na sequência de aminoácidos. As cepas de C. chauvoei isoladas no Brasil mostraram-se altamente conservadas em relação aos fatores de virulência avaliados neste estudo.

Generation and preliminary characterization of chimeric antibodies against neuraminidase of H7N9 subtype influenza A virus / 中华实验和临床病毒学杂志

Objective@#To develop the chimeric antibodies against neuraminidase (NA) of H7N9 and to identify their biological activity and function.@*Methods@#The genes of variable regions of the light chain (VL) and heavy chain (VH) obtained by mouse hybridoma technology were cloned respectively into the expression VH and VL vectors bearing human-derived Cγ1, and Cκ1 and co-transfected into 293T cells. The chimeric antibodies were purified and their functions were investigated.@*Results@#Two chimeric antibodies, 1E2 and 3E3 against neuraminidase (NA) of H7N9 were obtained. Both antibodies recognized similar antigenic epitopes. MAb 1E2 and 3E3 could prevent the infectivity with H7N9 and H11N9 virus and reduce their size of viral plaque.@*Conclusions@#The chimeric antibodies specific for N9 could prevent the infection of N9 subtype influenza virus as well as the NAI-resistant mutants and could be a potential immunotherapy approach for H7N9 treatment.

Neuraminidase Inhibitors from the Fruiting Body of Glaziella splendens

Neuraminidase (NA) cleaves the glycosidic bond linkages of sialic acids to release the mature virions from infected cells and has been an attractive therapeutic target for anti-influenza agents. In our ongoing investigation of NA inhibitors in mushroom extracts, we found that the extract the fruiting body of Glaziella splendens potently inhibited neuraminidase. The fruiting bodies of G. splendens were extracted and partitioned successively with hexane, ethyl acetate, and butanol. The ethyl acetate soluble-layer was subjected to silica gel and Sephadex LH-20 column chromatographies, and MPLC to obtain five compounds (1–5). Their structures were determined by spectroscopic methods. NA inhibitory activity of these compounds was evaluated using NAs from recombinant rvH1N1, H3N2, and H5N1 influenza A viruses. One compound (1) was elucidated as a new azaphilone derivative, and four compounds (2–5) were identified as entonaemin A, comazaphilone D, rubiginosin A, and entonaemin B, respectively. Compounds 3 and 4 showed considerable inhibitory activity against three types of neuraminidases with the IC₅₀ values of 30.9, 41.8, and 35.7 µM for 3 and 46.5, 50.4, and 29.9 µM for 4, respectively. This study reveals that the fruiting bodies of G. splendens possess azaphilone derivatives with the NA inhibitory activity. This is the first report on the isolation of neuraminidase inhibitors from the fruiting bodies of G. splendens.

Effect of plasma membrane-associated sialidase NEU3 activity on the proliferation and apoptosis of osteosarcoma MG-63 cells / 国际肿瘤学杂志

Objective To investigate the effect of plasma membrane-associated sialidase 3(NEU3) activity on the proliferation and apoptosis of osteosarcoma MG-63 cells in vitro. Methods MG-63 cells were cultured in vitro. Anti-NEU3 antibody(Ab)immunofluorescent staining was used to indicate the cellular locali-zation of NEU3 in MG-63 cells. The cells treated with 0 nmol/ L 2-deoxy-2,3-didehydro-N-acetyl neuraminic acid(DANA)or 0 μg/ ml anti-NEU3 Ab were used as blank control groups. The cells were treated with 10, 20,50 nmol/ L DANA,or 0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 24 h or 48 h,respectively. The inhibition rates of the cell proliferation and cell apoptosis rates were measured with CCK-8 and flow cytometry. The expression levels of oncogene-related proteins,Ras protein and Bcl-2 protein,were detected by Western blotting. Results The immunofluorescence result showed that NEU3 was located in the cytoplasm of MG-63 cell. After treating with 0,10,20,50 nmol/ L DANA for 48 h,the inhibition rates of cell proliferation were 0, 15. 10％ ± 3. 23％ ,41. 46％ ± 2. 31％ ,64. 68％ ± 4. 12％ ,with significant statistical difference(F = 99. 90, P < 0. 001),and the following contrast between each two groups met the statistical significance(all P < 0. 05). After treating with 0,0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 48 h,the inhibition rates of cell proliferation were 0,9. 34％ ± 1. 53％ ,19. 66％ ± 4. 18％ ,42. 50％ ± 5. 68％ ,and the difference was statistically signifi-cant(F = 25. 67,P < 0. 001),and the following contrast between each two groups met the statistical signifi-cance(P < 0. 05),except the difference between 0. 5 and 1. 0 μg/ ml groups(P > 0. 05). When the MG-63 cells were treated with 0,10,20,50 nmol/ L DANA for 24 h,the cell apoptosis rates were 4. 05％ ± 0. 07％ , 4. 15％ ± 0. 23％ ,12. 85％ ± 1. 48％ ,8. 29％ ± 0. 86％ ,respectively,and the difference was statistically sig-nificant(F = 23. 21,P < 0. 001). And the following contrast between each two groups met the statistical signi-ficance(P < 0. 05),except the differences between 0 nmol/ L and 10 nmol/ L,20 nmol/ L and 50 nmol/ L groups(P > 0. 05). When the MG-63 cells were treated with 0,0. 5,1. 0,2. 0 μg/ ml anti-NEU3 Ab for 24 h,the cell apoptosis rates were 4. 05％ ± 0. 07％ ,20. 13％ ± 2. 97％ ,20. 29％ ± 2. 82％ ,20. 58％ ± 0. 70％ ,with statistical significant difference(F = 15. 36,P = 0. 001). And the following contrast between each two groups showed that the differences between 0 μg/ ml and each treated group were statistically signifi-cant(P < 0. 05),while the differences between two treated groups were not statistically significant( P >0. 05). Western blotting results showed that the expression levels of Ras and Bcl-2 decreased with the increasing concentrations of DANA and anti-NEU3. Conclusion Inhibition of NEU3 enzyme activity can suppress the survival rate of MG63 cells and increase the cell apoptosis. The possible mechanism may be related to the declined expression of oncogene-related proteins Ras and Bcl-2,which suggests that NEU3 may be a possible target for treating osteosarcoma.

Resistance of influenza virus in Henan Province to neuraminidase inhibitor from 2017 to 2018 / 中华疾病控制杂志

Objective To analyze the resistance of influenza virus to neuraminidase inhibitors (NAI) in Henan province during 2017-2018. Methods Virus were collected from the Henan Influenza Surveillance Network during 2017-2018. 36 confirmed influenza virus(with 15 H1pdm09，6 H3N2 and 15 B) were selected to test resistance to oseltamivir and zanamivi with fluorescence(FL). The NAI sensitive reference viruses were A/California/12/2012(H1pdm09)-275H,A/Beijing Haidian/1942/2014(H3N2)-119E and B/Rochester/02/2001-198D. The NAI resistant reference viruses were A/ Texas/23/2012(H1pdm9)-H275Y, A/Texas/12/2007(H3N2)-E119V and B/Rochester/02/2001-D198N. Results The half maximal inhibitory concentration(IC50) of A/California/12/2012(H1pdm09)-275H, A/Beijing-Haidian/1942/2014(H3N2)-119E and B/Rochester/02/2001-198D for oseltamivir were 0.29 nmol/L (nM),0.10 nM and 12.71 nM, and for zanamivir were 0.2 nM, 0.49 nM and 0.33 nM respectively. The IC50 for oxastatin of H1pdm09 and H3N2 ranged from (0.28-1.37 nM) and (0.08-0.17 nM) respectively, the IC50 for zanamivir ranged from (0.15-0.49 nM) and (0.12-0.22 nM), all was within 10 fold IC50 of the reference virus(corresponding type); the IC50 value of type B for oseltamivir and zanamivir ranged from (11.83-24.59 nM) and (0.48-1.25 nM), all was within 5 fold IC50 of the reference virus. Conclusion All the tested influenza strains isolated in Henan province during 2017-2018 were sensitive to NAI.