FluClass: A novel algorithm and approach to score and visualize the phylogeny of the influenza virus using mass spectrometry

A novel computer algorithm FluClass has been developed to facilitate the phylogenetic classification of influenza virus using mass spectral data. FluClass accepts a DNA or protein-based phylogenetic tree as input and generates theoretical peptide mass lists for each node. An experimental mass spectrum from an influenza virus protein digest is then placed onto the phylogenetic tree using a novel random resampling function (Z-score) that allows the scoring of spectrum against both internal and leaf nodes. Testing of the algorithm using hemagglutinin protein sequences from human-host influenza viruses showed that the Z-score performs comparably to the Profound scoring method for the scoring of leaf nodes and is substantially better at scoring internal nodes. Scoring of internal nodes allows colorizations of nodes of the phylogenetic tree enabling the classification of the query spectrum to be rapidly visualized. Finally we demonstrate the utility of FluClass on experimental spectra from six strains. Given that mass spectrometry data can be generated rapidly for influenza virus proteins, FluClass provides a fast and direct method for phylogenetic analysis of influenza proteins.     

Pharmacophore Mode lingand Virtual Screening to Design the Potential Influenza Virus Endonuclease Inhibitors

Influenza virus endonuclease is an attractive target for antiviral therapy in the treatment of influenza infection. The purpos e of this study is to design a novel antiviral agent with improved biological activities against the influenza virus endonuclease. In this study, chemical feature‐based 3D pharmacophore models were developed from 41 known influenza virus endonuclease inhibitors. The best quantitative pharmacohore model (Hypo 1), which consists of two hydrogen‐bond acceptors and two hydrophobic features, yields the highest correlation coefficient (R = 0.886). Hypo 1 was further validated by the cross validation method and the test set compounds. The application of this model for predicting the activities of 11 known influenza virus endonuclease inhibitors in the test set shows great success. The correlation coefficient of 0.942 and a cross validation of 95;% confidence level prove that this model is reliable in identifying structurally diverse compounds for influenza virus endonuclease inhibition. The most active compound (compound 1) from the training set was docked into the active site of the influenza virus endonuclease as an additional verification that the pharmacophore model is accurate. The docked conformation showed important hydrogen bond interactions between the compound and two amino acids, Lys 134 and Lys 137. After validation, this model was used to screen the NCI chemical database to identify new influenza virus endonuclease inhibitors. Our study shows that the to pranking compound out of the 10 newly identified compounds using fit value ranking has an estimated activity of 0.049 μM. These newly identified lead compounds can be further experimentally validated using in vitro techniques.     

禽流感病毒HA蛋白与人受体的分子对接

血凝素(hemagglutinin,HA)是位于禽流感病毒表面的糖蛋白。在病毒感染过程中,HA与禽类宿主细胞表面受体结合,介导病毒膜与宿主核内体膜的融合,在传染过程中发挥关键作用。自然界中的禽流感病毒处于不断演化之中,其HA的禽受体结合位点常常发生氨基酸变异。因此,当HA变异体与人受体结合能力较强时,禽流感病毒往往会发生跨种传播而感染人。为预防禽流感的跨种传播,人们迫切需要发展大规模快速检测或预测HA变异体与人受体结合亲和力的方法,以评估各种新发禽流感病毒的跨种传播能力,提前筛选出有潜在危险的病毒株。针对此问题,本研究以H7N9亚型的HA蛋白H7为研究对象,发展了一种运用分子对接的计算方法,预测HA变异体与人受体的结合亲和力。该方法的计算结果表明,H7与人受体的结合亲和力普遍弱于有较强传染人能力的H1,说明H7N9亚型病毒的跨种传播能力普遍较弱;但是,计算分析也揭示,部分新发的H7N9毒株的HA有强的人受体结合亲和力,提示在自然演化过程中,H7N9病毒有可能演化出具有较强的感染人能力的新毒株,这与2013年禽流感疫情的实际发生情况相一致。因此,本文所发展的计算方法可用于快速预测新发禽流感病毒HA与人受体的结合亲和力,为新发禽流感病毒的跨种传播风险评估提供理论依据。     

Reduced surface area chromatography for flow-through purification of viruses and virus like particles

A method for flow-through purification of viruses and virus like nano-particles using a combination of binding and size-exclusion chromatography was developed. This technique relies on minimizing the external surface area per unit volume available for virus binding by increasing the mean diameter of the beads used in the column. At the same time the impurity binding capacity of the column is maximized by utilizing beads with multiple functionalities of the optimum size. Purification of different types of viruses and virus-like-particles could be achieved using this technique. Flow-through purification of influenza virus using this technique yielded virus recoveries greater than 70-80% coupled with impurity removal greater than 80%. Finally an approach to optimize and facilitate process development using this technology is presented. Since the impurity binding occurs via a non-specific mechanism and virus recovery is achieved through reduced surface area, the technique is not limited to specific types of viruses and offers the potential as a universal purification tool.     

糖链在流感病毒侵袭细胞中的作用

介绍了细胞外表面上含唾液酸的糖链在流感病毒侵袭细胞中的作用以及糖链与流感病毒表面糖蛋白－血凝素结合的特异性。     

A network-based pharmacology study of active compounds and targets of Fritillaria thunbergii against influenza

Seasonal and pandemic influenza infections are serious threats to public health and the global economy. Since antigenic drift reduces the effectiveness of conventional therapies against the virus, herbal medicine has been proposed as an alternative. Fritillaria thunbergii (FT) have been traditionally used to treat airway inflammatory diseases such as coughs, bronchitis, pneumonia, and fever-based illnesses. Herein, we used a network pharmacology-based strategy to predict potential compounds from Fritillaria thunbergii (FT), target genes, and cellular pathways to better combat influenza and influenza-associated diseases. We identified five compounds, and 47 target genes using a compound-target network (C-T). Two compounds (beta-sitosterol and pelargonidin) and nine target genes (BCL2, CASP3, HSP90AA1, ICAM1, JUN, NOS2, PPARG, PTGS1, PTGS2) were identified using a compound-influenza disease target network (C-D). Protein-protein interaction (PPI) network was constructed and we identified eight proteins from nine target genes formed a network. The compound-disease-pathway network (C-D-P) revealed three classes of pathways linked to influenza: cancer, viral diseases, and inflammation. Taken together, our systems biology data from C-T, C-D, PPI and C-D-P networks predicted potent compounds from FT and new therapeutic targets and pathways involved in influenza.     

In silico structure-based design of enhanced peptide inhibitors targeting RNA polymerase PAN-PB1C interaction

Developing antivirals for influenza A virus (FluA) has become more challenging due to high range of antigenic mutation and increasing numbers of drug-resistant viruses. Finding a selective inhibitor to target highly conserved region of protein-protein interactions interface, thereby increasing its efficiency against drug resistant virus could be highly beneficial. In this study, we used in silico approach to derive FluAPep1 from highly conserved region, PA_N-PB1_C interface and generated 121 FluAPep1 analogues. Interestingly, we found that the FluAPep1 interaction region in the PA_N domain are highly conserved in many FluA subtypes. Especially, FluAPep1 targets two pandemic FluA strains, H1N1/avian/2009 and H3N2/Victoria/1975. All of these FluA subtypes PA_N domain (H1N1/H3N2CAN/H3N2VIC/H7N1/H7N2) were superimposed with PA_N domain from H17N10 and the calculated root mean standards deviations were less than 3 Å. FlexPepDock analysis revealed that FluAPep1 exhibited higher binding affinity (score -246.155) with the PA_N domain. In addition, around 86% of non-hot spot mutated peptides (FluAPep28-122) showed enhanced binding affinity with PA_N domain. ToxinPred analysis confirmed that designed peptides were non-toxic. Thus, FluAPep1 and its analogues has potential to be further developed into an antiviral treatment against FluA infection.     

Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin

Monoliths are chromatographic stationary phases, which were specially designed for efficient purification of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60 × 10¹⁰ viral particles/mL to at least 1.64 × 10¹¹ viral particles/mL, the limit of detection was found to be 2.56 × 10⁹ virus particles/mL and the limit of quantification was 5.12 × 10⁹ virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus.     

Screening of inhibitors for influenza A virus using high-performance affinity chromatography and combinatorial peptide libraries

The affinity inhibitor of fusion peptide of influenza A virus has been studied using a combination of high-performance affinity chromatography (HPAC) and combinatorial peptide libraries. Fusion peptide (FP) (1-11) of influenza A virus was used as the affinity ligand and immobilized onto the poly(glycidyl methacrylate) (PGMA) beads. Positional scanning peptide libraries based on antisense peptide strategy and extended peptide libraries were designed and synthesized. The screening was carried out at acidic pH (5.5) in order to imitate the environment of virus fusion. A hendecapeptide FHRKKGRGKHK was identified to have a strong affinity to the FP (1-11). The dissociation constant of the complex of the hendecapeptide and the FP (1-11) is 3.10 x 10(-6) mol l(-1) in a physiological buffer condition. The polypeptide has a fairly inhibitory effect on three different strains of influenza A virus H1N1 subtype.     

Immunogenicity and Efficacy of Liposome-encapsulated Influenza Split Vaccine in BALB/c Mice

The cellular immunity of current influenza split vaccine is relatively low. It is necessary to develop a novel vaccine to improve the cellular immunity. Thes of this study prepared liposome-encapsulated influenza split vaccine and tested it in BALB/c mice. The mice were immunized once with 4 μg of haemagglutinin of monovalent A/New Caledonia/20/99(H1N1) encapsulated with liposomes or the split virus vaccine only through intrastomach injection. In a comparative study, it was observed that the liposome-encapsulated vaccine elicited a higher neutralizing antibody response, more effectively stimulated spleen cell proliferation, increased cell subsets like CD4 and CD4 /CD8 , and triggered IL-4 and IFN-γ production.     

禽流感病毒H5N1病毒颗粒中鸡胚宿主蛋白的质谱分析

在A型流感病毒等许多包膜病毒的病毒颗粒中,除包含病毒基因组编码的结构蛋白外,还包含来源于宿主细胞的多种蛋白。然而,在鸡胚内增殖的病毒颗粒所包含的宿主蛋白的种类尚不清楚。本研究采用20%～60%(w/w)蔗糖密度梯度离心法分离纯化了繁殖于鸡胚的流感病毒,并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)结合质谱法对纯化的流感病毒颗粒进行了全面的蛋白质组学分析。结果表明,在病毒颗粒中,除包含9种病毒编码蛋白外,还发现了12种来源于鸡胚的蛋白,如膜联蛋白A2、肽酰脯氨酰顺反异构酶B、过氧化物酶1、磷酸甘油酸激酶、丙酮酸激酶肌组织异构酶等,以及2种细胞骨架蛋白(微管蛋白b-3和肌动蛋白)。     

GC/MS-based metabolomics reveals fatty acid biosynthesis and cholesterol metabolism in cell lines infected with influenza A virus

Metabolomics is the downstream of systems biology and has drawn significant interest for studying the metabolic networks from cells to organisms. To profile the metabolites in two different cell lines (A549 and AGS) infected with influenza A virus, gas chromatography coupled with mass spectrometry (GC/MS) was employed. Some differentiating metabolites in the cell lines were tentatively identified using reference library, interpreted and visualized by applying principal components analysis (PCA) and cluster heat map. Consequently, metabolic flux profiling allowed the differentiation of fatty acid biosynthesis and cholesterol metabolism during viral replication in the cell lines. The change in fatty acid turnover was also observed. Metabolomics investigation also revealed the different responses between A549 and AGS cell lines to the virus infection. From the pattern recognition results, AGS cell line might be more susceptible to influenza A virus. Regarding the fact that AGS is a poorly differentiated gastric adenocarcinoma cell line whereas A549 is a relatively differentiated lung tumor one, it is speculated that viral replication might be associated with the cell differentiations.     

利用网络药理学方法研究热毒宁注射液抗流感病毒的分子作用机制

采用分子对接、网络分析预测热毒宁注射液抗流感病毒的分子作用机制, 并通过已建立的体外流感病毒神经氨酸酶筛选模型对网络预测结果验证. 结果表明, 热毒宁注射液所含化合物在化学空间上具有类药性质; 网络分析揭示出热毒宁注射液是通过与流感病毒吸附、脱壳、复制以及释放等环节的多个蛋白相互作用发挥抗流感病毒作用的; 对于预测的15 个活性分子而言, 实验结果初步证实, 对A型流感病毒, 木犀草素呈现较强的抑制作用, 槲皮素则呈现较弱的抑制作用, 这也初步证实了预测结果.     

Synthesis and Biological Evaluation of Analogs of Methyl Ursolate 3-O-β-Chacotrioside as H5N1 Viral Entry Inhibitors

The earliest stage of influenza virus infection is the viral entry to the host cell. In our previous work, we discovered the first three small molecule H5N1 viral entry inhibitors 1–3. Here, based on saponin 3, methyl ursolate 3-O-β-chacotrioside, several analogs were synthesized and evaluated to understand the structure-activity relationships of this type of compound on the H5N1 viral entry inhibitory activity. The preliminary studies demonstrated that unlike saponins 1 and 2, it is possible to reduce the 3-O-chacotriosyl residue of compound 3 to a disaccharide without affecting the viral entry activities significantly. The results obtained will render new clues to the understanding of the antiviral profile for these types of compounds.     

Testing of the influenza virus purification by CIEF

In virological practice, the pre‐concentration, purification and subsequent determination of the purity and concentration of the viruses from the cultural medium and/or from the real sample are required. The conventional techniques used today are equipment demanding, time‐consuming and laborious. In this study, the CIEF of influenza viruses with UV detection has been developed and subsequently used to test the purification of the virus from the biological samples. The equine and swine influenza viruses present in infected allantoic fluid of specific pathogen free embryonated chicken eggs were precipitated by using PEG 6000 and sodium chloride. The precipitated viruses were centrifuged at 14 000×g, and the impurities of different densities were removed by using the sucrose gradients. The efficiency of the virus purification technique was examined by the CIEF and compared to the results of real‐time PCR. The pIs of both influenza viruses were determined. Simultaneously, the CIEF was found to be a suitable method for the rapid testing of the efficiency of the virus purification.     

Detection of influenza virus: traditional approaches and development of biosensors

Influenza is an acute respiratory disease caused by the influenza virus. The disease occurs annually, causing fatality in the elderly and children and billions of dollars loss in business and productivity. Traditional viral detection methods include MDCK cell culture, complement fixation, hemagglutinin-inhibition, and recently RT-PCR. Although effective, these methods generally involve labor-intensive laboratory procedures and often require trained personnel to carry them out. The development of biosensor technologies will enable rapid and specific disease diagnosis on-site so that a clinician can quickly determine whether treatment is needed. This paper reviews traditional viral assays and progress in the biosensor development for influenza virus. Recent advances in single-step direct detection using non-labeling techniques such as surface plasmon resonance, quartz-crystal microbalance, and colorimetric functional polymers are discussed.     

液相色谱-质谱联用分析流感病毒感染引起宿主蛋白类泛素化修饰

干扰素刺激基因15编码蛋白质(Interferon stimulated gene 15 kDa protein, ISG15)是最早被鉴定的类泛素分子蛋白质,在病毒感染和免疫调节等方面具有重要作用。本研究利用免疫沉淀技术将被类泛素 ISG15修饰的蛋白富集纯化,采用液相色谱-质谱联用技术对流感病毒感染 A549宿主细胞过程中产生的类泛素 ISG15修饰蛋白进行了分析。实验结果表明,在流感病毒感染的实验组 A549细胞中,鉴定到了22种来源于宿主细胞的ISG15修饰的蛋白,包括类泛素蛋白 ISG15、细胞周期蛋白-T1、热休克蛋白71、钙调素结合蛋白、真核翻译起始因子等,以及1种来源于流感病毒的非结构蛋白 NS1。在鉴定的22种宿主蛋白中,有6种蛋白在未感染病毒的对照组 A549细胞中也得到鉴定,包括膜联蛋白 A1、果糖二磷酸醛缩酶 A、线粒体三磷酸腺苷合成酶亚基 g、烯醇化酶、肌动蛋白、微管蛋白。生物信息学分析表明,流感病毒感染引起的 ISG15修饰的宿主蛋白分别归属于9个不同的蛋白分类,包括细胞骨架蛋白、分子伴侣蛋白、酶调节剂、核酸结合蛋白、激酶类、转移酶类、转录因子、氧化还原酶类以及结构蛋白。本研究为大规模分析鉴定 ISG15修饰蛋白提供了一种特异、有效的研究方法。     

单管高灵敏度等温扩增技术快速检测甲型H1N1流感病毒

建立了单管逆转录环介导等温扩增法(RT-LAMP)快速检测甲型H1N1流感病毒的方法.针对甲型H1N1流感病毒的M基因和HA基因的保守区,设计了两组特异性引物,分别用于筛选甲型流感病毒及鉴定甲型H1N1流感病毒.对反应体系中的关键因素进行优化,反应结果可直接通过浊度或者SYBR Green Ⅰ荧光进行判定.本方法最低可...